WO2022183531A1 - 一种可跨种裂解的黄单胞菌噬菌体和其组合物、试剂盒和应用 - Google Patents

一种可跨种裂解的黄单胞菌噬菌体和其组合物、试剂盒和应用 Download PDF

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WO2022183531A1
WO2022183531A1 PCT/CN2021/081420 CN2021081420W WO2022183531A1 WO 2022183531 A1 WO2022183531 A1 WO 2022183531A1 CN 2021081420 W CN2021081420 W CN 2021081420W WO 2022183531 A1 WO2022183531 A1 WO 2022183531A1
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xanthomonas
phage
gj19p1
composition
xanthomonas phage
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French (fr)
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肖逍
丁良
丛郁
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菲吉乐科(南京)生物科技有限公司
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    • 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
    • A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
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    • A01N47/10—Carbamic acid derivatives, i.e. containing the group —O—CO—N<; Thio analogues thereof
    • A01N47/12—Carbamic acid derivatives, i.e. containing the group —O—CO—N<; Thio analogues thereof containing a —O—CO—N< group, or a thio analogue thereof, neither directly attached to a ring nor the nitrogen atom being a member of a heterocyclic ring
    • A01N47/14—Di-thio analogues 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
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    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10211—Podoviridae
    • C12N2795/10221—Viruses as such, e.g. new isolates, mutants or their genomic sequences
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    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10211—Podoviridae
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    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10211—Podoviridae
    • C12N2795/10232—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
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    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the field of bacteriophage, and more particularly, to a Xanthomonas bacteriophage that can be split across species and its composition, kit and application.
  • Xanthomonas straight rod-shaped bacteria, terminal flagellar motility, obligate aerobic.
  • All Xanthomonas species are phytopathogens and can cause plant diseases.
  • a total of 14 pathogenic species in the genus Xanthomonas have been included in the "People's Republic of China Entry Phytosanitary Pest List" due to their important quarantine significance, including: sugarcane white streak (Xanthomonas albilineans), banana necrosis Streak (Xanthomonas arboricola pv.celebensis), Xanthomonas axonopodis pv.betlicola, Xanthomonas axonopodis pv.citri, Xanthomonas axonopodis pv.citri Xanthomonas axonopodis pv.manihotis, Xanthomonas axonopodis pv.vasculorum, Xanthomonas axonopodis pv.mangiferaeindicae , Xanthomonas campestris pv.mangiferaeindicae
  • Bacteriophages are a class of viruses that specifically lyse bacteria.
  • the main chemical components are composed of proteins and nucleic acids. They are widely present in soil, air, water and living organisms. They have strong specificity and bind to specific sites on the surface of bacterial cells. Viruses that kill specific bacteria afterward. Since the first discovery of bacteriophages by Frederik Tword in 1915, a growing body of research has demonstrated that bacteriophages have high antimicrobial activity and specificity against drug-resistant bacteria and prevent damage to microbial communities. Compared with antibiotic treatment, it has extremely low side effects and is more rapid and effective, does not inhibit the body's natural immunity or cause allergic reactions, and is not invasive and toxic to humans, other mammals and plants. However, the current research on bacteriophage only focuses on the screening and biological characteristics, and there are few reports on its application. Therefore, screening and applying suitable potent phages is an effective way to develop new antibacterial agents.
  • the present application provides a cross-species lysable Xanthomonas phage and its composition, kit and application.
  • the present invention provides a Xanthomonas phage that can be split across species, using the following technical scheme:
  • a Xanthomonas phage that can be split across species the Xanthomonas phage is Xanthomonas phage GJ19P1 (Xanthomonas phage GJ19P1), and the deposit number is CCTCC NO: M 2020805.
  • the present application provides a Xanthomonas phage GJ19P1, which has excellent pH resistance and has been deposited in the China Type Culture Collection for preservation, and the preservation number is CCTCC NO: M 2020805.
  • 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 Xanthomonas phage of the present invention only needs to add a small amount to infect Xanthomonas 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 Xanthomonas bacteriophages of the present invention can all play a good preventive and control effect.
  • the Xanthomonas phage GJ19P1 of this scheme can be used to prevent and treat bacterial diseases caused by a variety of Xanthomonas pathogenic species.
  • the titer of the Xanthomonas phage GJ19P1 is reduced by no more than 2 orders of magnitude after 8 hours of ultraviolet radiation.
  • the Xanthomonas phage GJ19P1 of the present invention has good tolerance, the titer decreases less, and can play a better control effect on pathogenic bacteria.
  • the Xanthomonas phage GJ19P1 (Xanthomonas phage GJ19P1) can efficiently identify different Xanthomonas pathogenic species and has a wide lysis range.
  • the Xanthomonas phage GJ19P1 of the present invention has strong lysis ability to 9 different Xanthomonas pathogenic strains, and its lysis rate can reach 91.8%; The lysis ability of bacterial pathogenic varieties is above 85%.
  • the Xanthomonas phage GJ19P1 of the present invention can be used for preventing and treating bacterial diseases caused by various Xanthomonas pathogenic species.
  • the present invention provides a composition of Xanthomonas phage that can be split across species, using the following technical scheme:
  • composition of Xanthomonas phage that can be split across species contains at least one Xanthomonas phage GJ19P1 (Xanthomonas phage GJ19P1).
  • the composition includes a chemical bactericide.
  • the chemical fungicide is Dysenium 700 times liquid.
  • the Xanthomonas phage GJ19P1 and the chemical bactericide are used in combination as a composition.
  • the proportional relationship between the Xanthomonas phage GJ19P1 and the 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 knowledge in the art.
  • the composition comprises Xanthomonas axonopodis phage YHC5 (Xanthomonas axonopodis phage YHC5), the deposit number is CCTCC NO: M 2018579; Actinidiae phagePSA-P1), the deposit number is CCTCC NO: M 2020252; Ralstonia solanacearum phage GP3 (Ralstonia solanacearum phageGP3), the deposit number is CCTCC NO: M2016635.
  • the Xanthomonas phage GJ19P1 (Xanthomonas phage GJ19P1) can also be used in combination with other phages, and its composition can achieve a lysis rate of more than 97% against Xanthomonas rugosa, and has stronger lysis properties. .
  • the present invention provides a kit of Xanthomonas phage that can be split across species, using the following technical scheme:
  • kits of Xanthomonas bacteriophages that can be split across species, the kit is provided with a combination of the Xanthomonas bacteriophage GJ19P1 (Xanthomonas phage GJ19P1) or the Xanthomonas bacteriophage GJ19P1 (Xanthomonas phage GJ19P1) thing.
  • the Xanthomonas phage of the present invention is applied to the rapid detection of Xanthomonas, including but not limited to the detection of Xanthomonas in the form of test strips, test paper boxes, etc., or the detection of targets in clinical samples.
  • the pathogenic bacteria are screened to effectively ensure the sensitivity of the detection.
  • the present invention provides a kind of application of the Xanthomonas phage composition that can be split across species, adopts the following technical scheme:
  • the composition of the Xanthomonas bacteriophage GJ19P1 is used as an active ingredient of a biological disinfectant or a biological pesticide, preventing but not limited to by yellow Bacterial disease caused by the bacterium.
  • the Xanthomonas phage GJ19P1 and its composition can be used for the treatment and prevention of bacterial infections caused by Xanthomonas, but not limited to, Xanthomonas, and used for the prevention and treatment of Xanthomonas, And not limited to biological agents for diseases caused by Xanthomonas.
  • the present invention has the following beneficial effects:
  • the Xanthomonas phage GJ19P1 of the present invention has higher tolerance to ultraviolet rays and acidic environments, is suitable for different control environments, and can play a better biological control effect on citrus canker;
  • the Xanthomonas bacteriophage GJ19P1 of the present invention is a potent bacteriophage isolated from nature, the test bacteriophage does not contain virulence genes or bad genes, the DNA of the bacteriophage cannot encode proteins that may cause potential health risks, and there is no carrier the possibility of lysogenic genes;
  • the Xanthomonas phage GJ19P1 of the present invention has high affinity and lysis ability, and can reach a titer of more than 10 10 PFU/mL within 24 hours of culture; the Xanthomonas phage GJ19P1 can specifically partially or completely kill the bacteria Live Xanthomonas only needs a small amount of initial phage to complete a large number of proliferations, providing a source of high-quality phage strains for the industrial production of phage fungicides; those skilled in the art can use the descriptions in this application and common knowledge in the field. Monospora phage GJ19P1 or its composition is prepared into various products applied in detection, disinfection and plant protection, etc. and industrially applied;
  • the Xanthomonas phage GJ19P1 of the present invention is a strict virulent phage, which is highly specific and lytic to the host bacteria, and has a wide host range, and is resistant to 9 species of 184 different Xanthomonas pathogenic species.
  • Xanthomonas phage GJ19P1 can be used as an active ingredient for various products used in environmental disinfection, such as but not It is limited to disinfection and decontamination of water distribution systems, irrigation facilities, aquaculture facilities, public and private facilities or other environmental surfaces in the form of liquid immersion, spraying, and combined use with aqueous carriers, which 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 carriers include but are not limited to phosphate buffer, TSB medium, LB medium, chlorine free water, etc.;
  • the Xanthomonas phage GJ19P1 of the present invention could not identify any of the 10 non-host pathogenic bacteria tested, indicating that its specificity was good;
  • the Xanthomonas phage GJ19P1 of the present invention can be used for the preparation of compositions, reagents or kits for rapid detection of Xanthomonas, including but not limited to the detection of Xanthomonas in target samples in the form of test paper and kits.
  • the detection of single bacterium, or the screening of target pathogenic bacteria in clinical samples, can effectively ensure the sensitivity of the detection;
  • Xanthomonas phage GJ19P1 and its composition have good killing ability to Xanthomonas with a concentration of 10 3 PFU/mL in liquid medium; when the concentration of Xanthomonas phage GJ19P1 is greater than or equal to 10 4 PFU/mL When it is used, the killing rate of Xanthomonas is over 98.2%, and it has no antagonistic effect on other combined substances;
  • the Xanthomonas phage GJ19P1 (Xanthomonas phage GJ19P1) of the present invention and its composition can be prepared by those skilled in the art according to the records of the present application and common knowledge in the art, and can be used to prevent and control Xanthomonas phage, and is not limited to Xanthomonas phage GJ19P1. Biopharmaceuticals for diseases caused by monascus.
  • Figure 1 is a schematic diagram of plaques of Xanthomonas phage GJ19P1 (Xanthomonas phage GJ19P1).
  • Figure 2 is a schematic diagram of the morphological structure of Xanthomonas phage GJ19P1 (Xanthomonas phage GJ19P1) identified by electron microscopy under a transmission electron microscope.
  • Xanthomonas phage GJ19P1 (Xanthomonas phage GJ19P1), the deposit number is CCTCC NO: M 2020805, the deposit unit is the China Center for Type Culture Collection, and the deposit time is November 30, 2020;
  • Xanthomonas axonopodis phage YHC5 (Xanthomonas axonopodis phage YHC5), the deposit number is CCTCC NO: M 2018579, the deposit unit is the China Center for Type Culture Collection, and the deposit time is August 30, 2018.
  • Pseudomonas syringae pv. Actinidiae phagePSA-P1 Pseudomonas syringae pv. Actinidiae phagePSA-P1
  • the deposit number is CCTCC NO: M 2020252
  • the deposit unit is the China Center for Type Culture Collection
  • the deposit time is June 2020 30 days.
  • the deposit unit of Ralstonia solanacearum phage GP3 is China Type Culture Collection Center, the address is Wuhan University, Luojia Mountain, Wuchang, Wuhan City, Hubei province, zip code 430072; the deposit date is November 10, 2016 ; The deposit number is CCTCC NO: M2016635.
  • the citrus pathogenic variety of Xanthomonas axonopodis (Xanthomonas axonopodis pv.citri), the deposit number is ACCC 03526, which can be purchased from the depository.
  • Xanthomonas campestris pv.mangiferaeindicae with the deposit number of ATCC 11637, can be purchased by contacting the depository.
  • the Xanthomonas oryzae pv.oryzicola pathogenic variety (Xanthomonas oryzae pv.oryzicola), the deposit number is ACCC03523, can be purchased from the depository.
  • Pseudomonas syringae kiwifruit pathogenic varietal (Pseudomonassyringae pv. Actinidiae), the deposit number is ATCC BAA-2502, can be purchased by contacting the depository.
  • Ralstonia solanacearum the deposit number is ATCC11696D-5, 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/L Tris-HCl 50mL, gelatin 0.25g, distilled water 1000mL.
  • the source sample of the Xanthomonas phage GJ19P1 in the present invention is collected from sewage in Jiangning District, Nanjing City, Jiangsu province, filtered with double-layer filter paper, centrifuged at low speed at room temperature, and then filtered with a 0.22 ⁇ m filter membrane for the supernatant.
  • Xanthomonas phage GJ19P1 (Xanthomonas phage GJ19P1)
  • Xanthomonas phage GJ19P1 (Xanthomonas phage GJ19P1)
  • Xanthomonas phage GJ19P1 produced a single circular plaque with a translucent center and a diameter of 6-7 mm on the carpet grass Xanthomonas phage, referring to Figure 1.
  • Xanthomonas phage GJ19P1 (Xanthomonas phage GJ19P1), the deposit number is CCTCC M:2020805.
  • Example 1 Take the purified phage solution prepared in Example 1 for electron microscope observation: take 20 ⁇ L of the sample and drop it on the copper mesh, let it settle naturally for 15 minutes, use filter paper to absorb the excess liquid from the side, add 1 drop of 2% phosphotungstic acid on the copper mesh, and stain for 10 minutes , absorb the dye solution from the side with filter paper, and do electron microscope observation after drying.
  • Phage nucleic acid was extracted and sequenced using ⁇ phage genomic DNA kit. After nucleotide sequencing, the Xanthomonas phage GJ19P1 (Xanthomonas phage PSA-P1) has the nucleotide sequence shown in SEQ ID No.1.
  • the Xanthomonas phage GJ19P1 has a titer of more than 10 10 PFU/mL after culturing for 12 h.
  • Example 5 Deletion test of virulence gene or defective gene of Xanthomonas phage GJ19P1
  • 103 virulence genes identified as originating from lysogenic phages in pathogenic bacteria were selected as shown in Table 2. Whether it contains the following virulence genes.
  • Table 2 Whether it contains the following virulence genes.
  • the results show that Xanthomonas phage GJ19P1 does not contain the following virulence genes or bad genes, so it cannot encode proteins that may cause potential health risks, so Xanthomonas phage GJ19P1 will 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 Xanthomonas phage GJ19P1, the control group was given the same amount of normal saline for 15 days, and the experimental mice were sacrificed by severed neck, and the internal organs were examined.
  • the experimental results showed that this dose of Xanthomonas phage GJ19P1 had no effect on the daily behavior of mice. Anatomical examination of internal organs showed no abnormality.
  • the Xanthomonas phage GJ19P1 has biosafety and can be used as a crop disease control agent.
  • Example 7 Determination of the optimal multiplicity of infection (MOI) of Xanthomonas rugosa phage GJ19P1 against Xanthomonas rugosa
  • phage GJ19P1 purified solution prepared from Example 1
  • MOI purified phage solution effect
  • TSB liquid medium added to make the total volume of each tube the same.
  • the phage titer of each treatment was determined by the double-layer plate method. Each point was cultured in duplicate and averaged, and the MOI that produced the highest phage titer was the best multiplicity of infection. The experiment was repeated three times.
  • the Xanthomonas phage GJ19P1 (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 periodically sampled to detect its titer.
  • Xanthomonas rugosa phage GJ19P1 (prepared by Example 7) with a titer of 1 ⁇ 10 8 PFU/mL, spread it in a 90 mm sterile petri dish, and put it into an ultra-clean workbench. Irradiated under UV lamp (20w, 20cm). 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.
  • the results are shown in Table 8.
  • the titer of the phage GJ19P1 of Xanthomonas rugosa decreased by 2 orders of magnitude when irradiated with ultraviolet rays for 8 hours. Therefore, the phage of the present invention is more resistant to ultraviolet rays.
  • Phage lysis profiles were determined by double-layer plate spotting. Nine different Xanthomonas pathogenic strains were selected, totaling 184 strains. Pick the single colony of test strain respectively, inoculate it in the test tube filled with 3mL TSB liquid medium, and cultivate overnight at 180rpm at 28°C to obtain each bacterial strain. Take 500 ⁇ L of bacterial suspension mixed with TSB semi-solid agar medium and spread it on a common agar plate, take 5 ⁇ L of purified bacteriophage GJ19P1 solution (prepared from Example 7) and drop it on the plate. result.
  • Bacteriophage GJ19P1 has strong lysis ability to 9 species of 184 different Xanthomonas pathogenic strains, and its lysis rate can reach 91.8%; and it is pathogenic to the above single Xanthomonas.
  • the cracking capacity of the variants is above 85%.
  • Phage GJ19P1 has a broad lytic profile.
  • Example 11 Lysis test of Xanthomonas phage GJ19P1 to non-pathogenic beneficial bacteria Pick 5 non-pathogenic rhizobia, 5 non-pathogenic Bacillus subtilis were inoculated in 3 mL of TSB liquid medium respectively. In a test tube, culture at 180 rpm at 37° C. for 8 h to prepare bacterial strains of each strain. 300 ⁇ L of bacterial suspension was mixed with TSB semi-solid agar medium and spread on ordinary agar plates. 5 ⁇ L of purified bacteriophage GJ19P1 solution (prepared in Example 7) was dropped on the plate, and after natural air-drying, cultured at 37° C. for 12 h, and the results were observed.
  • Example 12 Bactericidal effect of Xanthomonas phage GJ19P1 in liquid
  • the control group was given Xanthomonas phage GJ19P1 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 Xanthomonas rugosa 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 Xanthomonas carpetgrass the number of colonies on the TSA plate ⁇ the dilution factor ⁇ 10.
  • composition 1 Take the stock solution of Xanthomonas phage GJ19P1 (Xanthomonas phage GJ19P1) and Carpetgrass Xanthomonas phage YHC5 (Xanthomonas axonopodis phage YHC5) with a titer of 2 ⁇ 10 9 PFU/mL, and mix the two phages in equal volume 1:1 evenly.
  • a 1:1 composition composition
  • composition 2 Take the stock solution of Xanthomonas phage GJ19P1 (Xanthomonas phage GJ19P1) and Pseudomonas syringae pv. Actinidiae phagePSA-P1 (Pseudomonas syringae pv.
  • the phage strains were uniformly mixed in equal volumes to make a 1:1 composition (composition 2).
  • composition 3 Take the stock solutions of 2 ⁇ 10 9 PFU/mL, Xanthomonas phage GJ19P1 (Xanthomonas phage GJ19P1), and Ralstonia solanacearum phage GP3 (Ralstonia solanacearum phageGP3), respectively, and mix the two strains of phages in an equal volume of 1:1. into a composition (composition 3).
  • composition 4 The titers were taken as 2x10 9 PFU/mL Xanthomonas phage GJ19P1 (Xanthomonas phage GJ19P1), carpet grass Xanthomonas phage YHC5 (Xanthomonas axonopodis phage YHC5) and Pseudomonas syringae kiwifruit pathogenic phage PSA-P1 (Pseudomonas syringae pv. Actinidiae phage PSA-P1) stock solution, three phages were uniformly mixed in equal volume 1:1:1 to prepare a composition (composition 4).
  • composition 5 The titers were respectively taken as 2 ⁇ 10 9 PFU/mL Xanthomonas phage GJ19P1 (Xanthomonas phage GJ19P1), carpet grass Xanthomonas phage YHC5 (Xanthomonas axonopodis phage YHC5) and Ralstonia solanacearum phage GP3 (Ralstonia solanacearum phage GP3) ), the three strains of phage were uniformly mixed in an equal volume of 1:1:1 to prepare a composition (composition 5).
  • composition 6 The titers were taken as 2 ⁇ 10 9 PFU/mL Xanthomonas phage GJ19P1 (Xanthomonas phage GJ19P1), Pseudomonas syringae pv. Actinidiae phagePSA-P1 (Pseudomonas syringae pv.
  • Ralstonia solanacearum phage GP3 Ralstonia solanacearum phageGP3
  • three phage strains were uniformly mixed in equal volume 1:1:1 to prepare a composition (composition 6).
  • the titers were taken as 2 ⁇ 10 9 PFU/mL Xanthomonas phage GJ19P1 (Xanthomonas phage GJ19P1), carpet grass Xanthomonas phage YHC5 (Xanthomonas axonopodis phage YHC5), Pseudomonas syringae kiwifruit pathogenic phage PSA-P1 (Pseudomonas syringae pv.Actinidiae phage PSA-P1) and the stock solution of Ralstonia solanacearum phage GP3 (Ralstonia solanacearum phage GP3), the 4 strains of phages were uniformly mixed in equal volume 1:1:1:1 to prepare a composition (combination item 7).
  • Example 14 Bactericidal effect of Xanthomonas phage GJ19P1 composition in liquid
  • each composition of the Xanthomonas phage GJ19P1 has a good killing effect on Xanthomonas rugosa. It shows that the Xanthomonas phage GJ19P1 can be used in combination with other substances to control bacteria, and has no antagonistic effect on other substances.
  • the combination of this embodiment is not limited to 700 times of dysenium solution, but also can be polydoxamycin, 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 other chemical disinfectants to achieve anti-kill effect.
  • Example 15 Prevention and control of citrus canker by Xanthomonas phage GJ19P1 and its composition Bacteriophage GJ19P1 (Xanthomonas phage GJ19P1) was uniformly mixed with equal volume of 700-fold dysenium solution (chemical bactericide) with a final concentration of 50% to prepare 1:1 composition 9, composition 10 and composition 11.
  • Example 16 Preparation and use of a kit of Xanthomonas phage GJ19P1 and its composition
  • the kit contains 5-10 mL of Xanthomonas phage GJ19P1 liquid or Xanthomonas with a titer of 1 ⁇ 10 7 PFU/mL Composition of bacteriophage GJ19P1, 1LTSB semi-solid medium, 1L TSA medium.
  • the use method of the kit is as follows: taking the composition of Xanthomonas phage GJ19P1 liquid or Xanthomonas phage GJ19P1 with a titer of 1 ⁇ 10 7 PFU/mL, and using the double-layer plate spotting method to measure the lysis profile of the test phage .
  • 300 ⁇ L of the bacterial suspension of the strain to be tested was mixed with 5 mL of TSB semi-solid medium and spread on a TSA plate. After natural air-drying, culture at the target temperature according to the growth characteristics of the strain to be detected, and observe the results.
  • the main component of the kit 1 is 5-10 mL of Xanthomonas phage GJ19P1 liquid with a titer of 3 ⁇ 10 8 PFU/mL.
  • 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 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 4 is 5-10 mL of Ralstonia solanacearum phage GP3 (Ralstonia solanacearum phage GP3) liquid with a titer of 3 ⁇ 10 8 PFU/mL.
  • the main component of the kit 5 is 5-10 mL, and the titer is a 700-fold solution of desenium with a final concentration of 50%.
  • the main components of the kit 6 are 5-10 mL of 3 ⁇ 10 8 PFU/mL of Carpetgrass Xanthomonas phage GJ19P1 liquid, and 5 to 10 mL of Carpetgrass Yellow with a potency of 3 ⁇ 10 8 PFU/mL Monosodium phage YHC5 liquid, 5-10 mL of 3 ⁇ 10 8 PFU/mL Pseudomonas syringae kiwifruit pathogenic variant phage PSA-P1 liquid, 5-10 mL of titer of 3 ⁇ 10 8 PFU/mL mL of R. solanacearum bacteriophage GP3 liquid, and 5 to 10 mL of 700-fold dysenium solution with a final concentration of 50%.
  • Test process 160 citrus trees were randomly divided into 8 groups (kit 1-kit 6, positive control group and negative control group) after adaptive cultivation for 1 month, with 20 trees in each group.
  • 1L of Xanthomonas rugosa citrus pathogenic varietal ACCC 03526 (Xanthomonas axonopodis pv.citri) with a final concentration of 1x10 3 cfu/mL was administered to the kit group 1 to kit group 6 and the positive control group by stem infusion method.
  • a total of 140 citrus trees were challenged, and 20 citrus trees in the negative control group were injected with 1 L of normal saline by stem infusion method.
  • the incidence of citrus in the kit 6 group is lower than that in the kit 1 group, indicating that the kit 6 containing the composition has a significant effect on the prevention and treatment of citrus canker, and the kit 6 containing the composition is more Kit 1 of Xanthomonas phage GJ19P1 was more effective in controlling the incidence of citrus.
  • Example 18 Application of the kit of Xanthomonas phage GJ19P1 and its composition to rice bacterial leaf spot
  • Kits 1 to 6 all used the kits of Example 17.
  • Test process 160 rice plants were randomly divided into 8 groups (kit 1-kit 6, positive control group and negative control group) after 1 month of adaptive culture, with 20 plants in each group.
  • 1 L of Xanthomonas oryzaes pv. oryzicola (Xanthomonas oryzaes pv.oryzicola) with a final concentration of 1 x 10 3 cfu/mL was irrigated to 140 strains from Kits 1 to 6 and the positive control group, respectively.
  • the rice plants were challenged, and 20 rice plants in the negative control group were treated with 1 L of normal saline by root irrigation.
  • the morbidity rate of rice plants in the kit 6 group was lower than that in the kit 1 group, indicating that the kit 6 containing the composition had significant control of rice bacterial leaf streak, and the kit containing the composition 6 is more effective than kit 1 containing only Xanthomonas phage GJ19P1 in controlling the incidence of rice plants.
  • Kits 1 to 6 all used the kits of Example 17.
  • Test process 160 cassava strains were randomly divided into 8 groups (kit 1 to kit 6, positive control group and negative control group) after adaptive cultivation for 1 month, with 20 plants in each group.
  • 1 L of Xanthomonas rugosa cassava wilt pathogenic varietal ACCC 03517 (Xanthomonas axonopodis pv. manihotis) with a final concentration of 1x10 3 cfu/mL was applied to Kits 1 to 6 and the positive control group with a total of 140
  • the cassava strains were challenged, and 20 cassava strains in the negative control group were injected with 1 L of normal saline by root irrigation.
  • kits 1 to 6 Three days after the challenge, 1 L of the thousand-fold dilutions of kits 1 to 6 were inoculated by root irrigation, and the positive control group and the negative control group were given the same amount of normal saline. The incidence of bacterial wilt disease of cassava within 15 days from inoculation was counted.
  • the incidence rate of cassava in the kit 6 group is lower than that in the kit 1 group, indicating that the kit 6 containing the composition has significant control of cassava bacterial wilt disease, and the kit 6 with the composition is more Kit 1 containing only Xanthomonas phage GJ19P1 was more effective in controlling the incidence of cassava.
  • Kits 1 to 6 all used the kits of Example 17.
  • Test process 160 mango trees were randomly divided into 8 groups (kit 1 to kit 6, positive control group and negative control group) after adaptive cultivation for 1 month, with 20 plants in each group. 1L of Xanthomonas campestris pv.mangiferaeindicae (Xanthomonas campestris pv.mangiferaeindicae) with a final concentration of 1x10 3 cfu/mL of kit 1 to kit 6 and the positive control group were treated by stem infusion method. 140 mango trees were challenged, and 20 mango trees in the negative control group were injected with 1 L of normal saline by stem infusion method.
  • the incidence rate of mangoes in the kit 6 group is lower than that in the kit 1 group, indicating that the kit 6 containing the composition has significant control of mango bacterial black spot, and the kit 6 containing the composition Compared with the kit 1 containing only Xanthomonas phage GJ19P1, it is more effective in controlling the morbidity.
  • Kits 1 to 6 all used the kits of Example 17.
  • Test process 160 kiwifruit trees were randomly divided into 8 groups (kit 1-kit 6, positive control group and negative control group) after adaptive cultivation for 1 month, with 20 trees in each group.
  • 1L of Pseudomonas syringae kiwifruit pathogenic variety ATCC BAA-2502 (Pseudomonassyringae pv. Actinidiae) with a final concentration of 1x10 3 cfu/mL was added to Kits 1 to 6 and the positive control group with a total of 140
  • One kiwifruit tree was challenged, and 20 kiwifruit trees in the negative control group were injected with 1 L of normal saline by stem infusion method.
  • the incidence of kiwifruit in the kit 6 group was lower than that in the kit 1 group, indicating that the kit 6 containing the composition had a significant effect on the prevention and treatment of kiwifruit canker, and the kit 6 containing the composition was more Kit 1 of Xanthomonas phage GJ19P1 was more effective in controlling the incidence.
  • Kits 1 to 6 all used the kits of Example 17.
  • Test process 160 ginger strains were randomly divided into 8 groups (kit 1 to kit 6, positive control group and negative control group) after adaptive cultivation for 1 month, with 20 strains in each group.
  • 1 L of Ralstonia solanacearum ATCC11696D-5 (Ralstonia solanacearum) with a final concentration of 1 x 10 3 cfu/mL was used to challenge 140 ginger strains in kits 1 to 6 and the positive control group by root irrigation.
  • 20 strains of ginger in the negative control group were injected with 1L of normal saline.
  • Three days after the challenge 1 L of the thousand-fold dilution of kit 1 to kit 6 were inoculated by injection, and the positive control group and the negative control group were given the same amount of normal saline.
  • the ginger morbidity rate of test kit 6 group is lower than that of test kit 1 group, indicating that the test kit 6 containing the composition is remarkable for the control of ginger and ginger blast, and the test kit 6 with the composition is more than only containing Kit 1 of Xanthomonas phage GJ19P1 was more effective in controlling the incidence.
  • Xanthomonas phage GJ19P1 Xanthomonas phage GJ19P1
  • its composition are highly safe, and can be used as preparation kits and active ingredients of biological disinfectants or biological pesticides. Bacterial diseases caused by the bacteria.

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Abstract

一种可跨种裂解的黄单胞菌噬菌体及其组合物、试剂盒和应用，所述地毯草黄单胞菌噬菌体为黄单胞菌噬菌体GJ19P1(Xanthomonas phage)，保藏编号为CCTCC NO:M 2020805；所述组合物中至少含有一株黄单胞菌噬菌体GJ19P1；所述试剂盒中带有黄单胞菌噬菌体GJ19P1或黄单胞菌噬菌体GJ19P1的组合物；所述黄单胞菌噬菌体GJ19P1的组合物用于但不限于杀死黄单胞菌。

Description

一种可跨种裂解的黄单胞菌噬菌体和其组合物、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，它涉及一种可跨种裂解的黄单胞菌噬菌体和其组合物、试剂盒和应用。
背景技术
黄单胞菌属，直杆状细菌，端生鞭毛运动，专性好氧。在培养基上可产生一种非水溶性的黄色色素，一种类胡萝卜素，其化学成分为溴芳基多烯，使菌落呈黄色。所有的黄单胞菌都是植物病原菌，可引起植物病害。
黄单胞菌属内共有14种致病变种因具有重要的检疫意义而被列入《中华人民共和国进境植物检疫性有害生物名录》，具体包括：甘蔗白色条纹病菌(Xanthomonas albilineans)、香蕉坏死条纹病菌(Xanthomonas arboricola pv.celebensis)、地毯草黄单胞菌胡椒致病变种(Xanthomonas axonopodis pv.betlicola)、地毯草黄单胞菌柑橘致病变种(Xanthomonas axonopodis pv.citri)、地毯草黄单胞菌木薯萎蔫致病变种(Xanthomonas axonopodis pv.manihotis)、地毯草黄单胞菌甘蔗致病变种(Xanthomonas axonopodis pv.vasculorum)、地毯草黄单胞菌芒果致病变种(Xanthomonas axonopodis pv.mangiferaeindicae)、野油菜黄单胞菌芒果致病变种(Xanthomonas campestris pv.mangiferaeindicae)、野油菜黄单胞菌香蕉致病变种(Xanthomonas campestris pv.musacearum)、木薯细菌性叶斑病菌(Xanthomonas cassavae)、草莓角斑病菌(Xanthomonas fragariae)、风信子黄腐病菌(Xanthomonas hyacinthi)、稻黄单胞菌稻白叶枯致病变种(Xanthomonas oryzae pv.oryzae)、稻黄单胞菌稻细条斑致病变种(Xanthomonas oryzae pv.oryzicola)和杨树细菌性溃疡病菌(Xanthomonas populi)。据中国检验检疫科学研究院动植物检疫信息资源共享服务平台的口岸截获数据统计显示，自2015年1月至2017年2月这两年间，我国各口岸分支局共在旅客携带物以及货物检疫中截获黄单胞菌共计586批次，涉及的货物大类有粮谷类、大豆类、蔬菜类、水果类、其他植物产品类、种苗类、原料及制品类、原木类等等。例如，稻黄单胞菌引起的白叶枯病是水稻生产中危害非常严重的细菌性病害之一。野油 菜黄单胞菌芒果致病变种引起的芒果细菌性黑斑病是芒果上一种常发性重要病害，该病严重影响芒果产量和果品商品价值。地毯草黄单胞菌柑橘致病变种是导致柑橘溃疡病的致病菌，严重影响柑橘果实的产量和品质。(林文力,肖伏莲.柑橘溃疡病菌检测方法及防治技术研究进展[J].现代农业科技,2012(22):112-114.)。
噬菌体是专门裂解细菌的一类病毒，主要化学成分由蛋白质和核酸组成，广泛存在于土壤、空气、水及生物体中，具有较强的专一性，其通过与细菌细胞表面特异位点结合后杀灭特异性细菌的病毒。自从Frederik Tword于1915年首次发现噬菌体以来，越来越多的研究证明了噬菌体具有较高的抗菌活性和专一性，可防治耐药性细菌并防止损伤微生物群落。与抗生素治疗相比，其副作用极低且更加快速有效，不会抑制机体的自然免疫或引起变态反应，同时对人类、其他哺乳动物以及植物不具有侵染性及毒性。然而目前对噬菌体的研究仅集中于筛选及生物特性的探讨，对其应用方面鲜有报道。因此，筛选并应用合适的烈性噬菌体，是研发新型抗菌制剂的一条有效途径。
目前，关于黄单胞菌噬菌体用于控制由黄单胞菌引起的植物细菌性病害相关研究甚少。
针对上述中的相关技术，发明人认为，急需寻找一种可跨种裂解的黄单胞菌噬菌体。
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，本申请提供一种可跨种裂解的黄单胞菌噬菌体及其组合物、试剂盒和应用。
第一方面，本发明提供一种可跨种裂解的黄单胞菌噬菌体，采用如下的技术方案：
一种可跨种裂解的黄单胞菌噬菌体，所述黄单胞菌噬菌体为黄单胞菌噬菌体GJ19P1(Xanthomonas phage GJ19P1)，保藏编号为CCTCC NO:M 2020805。
通过采用上述技术方案，本申请提供一种黄单胞菌噬菌体GJ19P1，该噬菌体具有优良的耐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 2020805。
优选的，所述黄单胞菌噬菌体GJ19P1(Xanthomonas phage GJ19P1)在感染复数MOI＝0.0001条件下培养24h，效价达到3.5×10 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。本发明的黄单胞菌噬菌体只需添加少量，即可侵染黄单胞菌并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。
优选的，所述黄单胞菌噬菌体GJ19P1(Xanthomonas phage GJ19P1)在pH＝2～10的条件下具有耐性，在96h内效价降低不超过4个数量级；黄单胞菌噬菌体GJ19P1(Xanthomonas phage GJ19P1)对9种不同黄单胞菌致病变种菌株均具有较强的裂解能力，裂解率可达91.8％；且黄单胞菌噬菌体GJ19P1(Xanthomonas phage GJ19P1)对单一黄单胞菌致病变种的裂解能力均在85％以上通过采用上述技术方案，可知在pH＝2时，即在酸性条件下，本发明的黄单胞菌噬菌体GJ19P1具有优异的耐性，当防治环境在pH＝2～10之间时，本发明的黄单胞菌噬菌体均可以起到良好的防治作用。本方案的黄单胞菌噬菌体GJ19P1可用于防治由多种黄单胞菌致病变种引起的细菌性病害。
优选的，所述黄单胞菌噬菌体GJ19P1(Xanthomonas phage GJ19P1)经紫外辐射8h后，效价降低不超过2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，本发明的黄单胞菌噬菌体GJ19P1具有良好的耐受性，效价降低较少，能够对病原菌起到较好的防治作用。
优选的，所述黄单胞菌噬菌体GJ19P1(Xanthomonas phage GJ19P1)可高效识别不同黄单胞菌致病变种，具有较广的裂解范围。
通过采用上述技术方案，本发明的黄单胞菌噬菌体GJ19P1对9种不同黄单胞菌致病变种菌株均具有较强的裂解能力，其裂解率可达91.8％；且其对单一黄单胞菌致病变种的裂解能力均在85％以上。本发明的黄单胞菌噬菌体GJ19P1可用于防治由多种黄单胞菌致病变种引起的细菌性病害。
第二方面，本发明提供一种可跨种裂解的黄单胞菌噬菌体的组合物，采用如下的技术方案：
一种可跨种裂解的黄单胞菌噬菌体的组合物，所述组合物中至少含有一株黄单胞菌噬菌体GJ19P1(Xanthomonas phage GJ19P1)。
优选的，所述组合物包括化学性杀菌剂。
优先的，所述化学性杀菌剂为代森铵700倍液。
通过采用上述技术方案，将所述的黄单胞菌噬菌体GJ19P1和化学性杀菌剂作为组合物联合使用。作为示例性的说明，黄单胞菌噬菌体GJ19P1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。
优先的，所述组合物包括地毯草黄单胞菌噬菌体YHC5(Xanthomonas axonopodis phage YHC5)，保藏编号为CCTCC NO:M 2018579；丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1(Pseudomonas syringae pv.Actinidiae phagePSA-P1)，保藏编号为CCTCC NO:M 2020252；茄科雷尔氏菌噬菌体GP3(Ralstonia solanacearum phageGP3)，保藏编号为CCTCC NO:M2016635。
通过采用上述技术方案，黄单胞菌噬菌体GJ19P1(Xanthomonas phage GJ19P1)还可与其它噬菌体混合使用，其组合物对地毯草黄单胞菌的裂解率可达97％以上，具有更强的裂解性。
第三方面，本发明提供一种可跨种裂解的黄单胞菌噬菌体的试剂盒，采用如下的技术方案：
一种可跨种裂解的黄单胞菌噬菌体的试剂盒，所述试剂盒中带有所述黄单胞菌噬菌体GJ19P1(Xanthomonas phage GJ19P1)或黄单胞菌噬菌体GJ19P1(Xanthomonas phage GJ19P1)的组合物。
通过上述技术方案，本发明的黄单胞菌噬菌体应用于黄单胞菌的快速检测，包括但不限于以试纸、试纸盒等形式对黄单胞菌进行检测，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，本发明提供一种可跨种裂解的黄单胞菌噬菌体组合物的应用，采用如下的技术方案：
一种可跨种裂解的黄单胞菌噬菌体组合物的应用，所述的黄单胞菌噬菌体GJ19P1的组合物，用作为生物类消毒剂或生物类农药的有效成分，防治但不限于由黄单胞菌引起的细菌性疾病。
通过采用上述技术方案，黄单胞菌噬菌体GJ19P1及其组合物可以用于治 疗和预防由黄单胞菌、且不限于由黄单胞菌引起的细菌感染，用作防治由黄单胞菌、且不限于由黄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、本发明的黄单胞菌噬菌体GJ19P1对紫外线、酸性环境具有较高的耐受性，适用于不同的防治环境中，能够对柑橘溃疡病起到较好的生物防治效果；
2、本发明的黄单胞菌噬菌体GJ19P1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、本发明的黄单胞菌噬菌体GJ19P1具有高度亲和性及裂解能力，在培养24h内可达到10 10PFU/mL以上的效价；黄单胞菌噬菌体GJ19P1可以特异性地部分或完全灭活黄单胞菌，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黄单胞菌噬菌体GJ19P1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、本发明的黄单胞菌噬菌体GJ19P1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9种184株不同黄单胞菌致病变种的裂解率高达91.8％；且对单一黄单胞菌致病变种的裂解能力均在85％以上；黄单胞菌噬菌体GJ19P1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、本发明的黄单胞菌噬菌体GJ19P1无法识别10株供试非宿主性致病性细菌中的任何一株，说明其特异性良好；
6、本发明的黄单胞菌噬菌体GJ19P1具有良好的稳定性，在pH＝2～10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2个数量级；
7、本发明的黄单胞菌噬菌体GJ19P1，可用于制备组合物、试剂或试剂盒，应用 于黄单胞菌的快速检测，包括但不限于以试纸、试剂盒等形式对目标样本中的黄单胞菌进行检测，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、黄单胞菌噬菌体GJ19P1及其组合物对液体培养基中浓度为10 3PFU/mL的黄单胞菌具有良好的杀灭能力；当黄单胞菌噬菌体GJ19P1浓度≥10 4PFU/mL时，其对黄单胞菌的杀灭率达98.2％以上，且对其他联用物质无拮抗作用；
9、本发明的黄单胞菌噬菌体GJ19P1(Xanthomonas phageGJ19P1)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黄单胞菌、且不限于由黄单胞菌引起的病害的生物药剂。
附图说明
图1是黄单胞菌噬菌体GJ19P1(Xanthomonas phageGJ19P1)的噬菌斑示意图。
图2是电镜鉴定的黄单胞菌噬菌体GJ19P1(Xanthomonas phage GJ19P1)透射电子显微镜下形态结构示意图。
具体实施方式
以下结合附图1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。
黄单胞菌噬菌体GJ19P1(Xanthomonas phage GJ19P1),保藏编号为CCTCC NO:M 2020805，保藏单位为中国典型培养物保藏中心，保藏时间为2020年11月30日；
地毯草黄单胞菌噬菌体YHC5(Xanthomonas axonopodis phage YHC5)，保藏编号为CCTCC NO:M 2018579，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。
丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1(Pseudomonas syringae pv.Actinidiae phagePSA-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。
茄科雷尔氏菌噬菌体GP3(Ralstonia solanacearum phage GP3)的保藏单位为中国典型培养物保藏中心，地址为湖北省武汉市武昌珞珈山武汉大学，邮编430072；保藏日期为2016年11月10日；保藏编号为CCTCC NO：M2016635。
地毯草黄单胞菌柑橘致病变种(Xanthomonas axonopodis pv.citri)，保藏编号为ACCC 03526，向保藏单位联系购买获得。
地毯草黄单胞菌木薯萎蔫致病变种(Xanthomonas axonopodis pv.manihotis)，保藏编号为ACCC 03517，可通过向保藏单位联系购买获得。
野油菜黄单胞菌芒果致病变种(Xanthomonas campestris pv.mangiferaeindicae)，保藏编号为ATCC 11637，可通过向保藏单位联系购买获得。
稻黄单胞菌稻细条斑致病变种(Xanthomonas oryzae pv.oryzicola)，保藏编号为ACCC03523，向保藏单位联系购买获得。
丁香假单孢菌猕猴桃致病变种(Pseudomonassyringae pv.Actinidiae)，保藏编号为ATCC BAA-2502，可通过向保藏单位联系购买获得。
茄科雷尔氏菌(Ralstonia solanacearum)，保藏编号为ATCC11696D-5，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/L Tris-HCl 50mL，明胶0.25g，蒸馏水1000mL。
实施例1 黄单胞菌噬菌体GJ19P1的分离制备及纯化培养
本发明中黄单胞菌噬菌体GJ19P1的来源样品采集于江苏省南京市江宁区污水，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GJ19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噬菌体宿主菌地毯草黄单胞菌GJ19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1mL SM液中室温下震荡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、45℃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50mL TSB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，即为黄单胞菌噬菌体GJ19P1(Xanthomonas phage GJ19P1)。黄单胞菌噬菌体GJ19P1(Xanthomonas phage GJ19P1)在地毯草黄单胞菌菌苔上产生单一的、中心透亮、直径为6～7mm的圆形噬菌斑，参照图1。黄单胞菌噬菌体GJ19P1(Xanthomonas phage GJ19P1)，保藏编号为CCTCC M:2020805。
实施例2 黄单胞菌噬菌体GJ19P1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黄单胞菌噬菌体GJ19P1形态发现， 该噬菌体在电子显微镜下呈现为1个直径约60nm的多面体立体对称的头部，及一个短收缩尾部。该噬菌体属于自复制短尾噬菌体科(Autographiviridae)。
实施例3 黄单胞菌噬菌体GJ19P1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，所述黄单胞菌噬菌体GJ19P1(Xanthomonas phage PSA-P1)具有SEQ ID No.1所示的核苷酸序列。
实施例4 黄单胞菌噬菌体GJ19P1效价的测定
用SM液做稀释液，将黄单胞菌噬菌体GJ19P1(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，黄单胞菌噬菌体GJ19P1培养12h后具有10 10PFU/mL以上的效价。
表1培养12h后黄单胞菌噬菌体GJ19P1的效价
培养时间 4h 8h 12h
噬菌体GJ19P1效价(PFU/mL) 5.2x10 9 2.8x10 10 4.6x10 10
实施例5 黄单胞菌噬菌体GJ19P1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，通过测定黄单胞菌噬菌体GJ19P1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，黄单胞菌噬菌体GJ19P1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，因此黄单胞菌噬菌体GJ19P1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 PCTCN2021081420-appb-000001
Figure PCTCN2021081420-appb-000002
Figure PCTCN2021081420-appb-000003
Figure PCTCN2021081420-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的黄单胞菌噬菌体GJ19P1，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，此剂量的黄单胞菌噬菌体GJ19P1对小鼠日常行为没有影响。解剖检查内脏未见异常。黄单胞菌噬菌体GJ19P1具有生物安全性，可作为农作物病害防治制剂。
实施例7 黄单胞菌噬菌体GJ19P1对地毯草黄单胞菌最佳感染复数(MOI)的测定
挑取宿主菌地毯草黄单胞菌GJ19单个菌落，接种到盛有3mL TSB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10mL TSB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GJ19P1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160rpm振荡培养12h。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不同感染复数下黄单胞菌噬菌体GJ19P1的效价
MOI GJ19P1(PFU/mL) 宿主菌(cfu/mL) GJ19P1效价(PFU/mL)
100 10 7 10 5 1.3×10 8
10 10 7 10 6 2.4×10 8
1 10 7 10 7 5.7×10 8
0.1 10 7 10 8 2.5×10 9
0.01 10 7 10 9 6.9×10 9
0.001 10 7 10 10 8.3×10 9
0.0001 10 6 10 10 3.5×10 10
0.00001 10 5 10 10 3.0×10 10
0.000001 10 4 10 10 2.4×10 10
0.0000001 10 3 10 10 1.5×10 10
由表3可知，培养12h条件下，噬菌体GJ19P1效价达到最高3.5×10 10PFU/mL时，其MOI＝0.0001。说明仅需少量初始黄单胞菌噬菌体GJ19P1，即可完成大量增殖。黄单胞菌噬菌体GJ19P1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 黄单胞菌噬菌体GJ19P1的pH和温度稳定性的测定
8-1：不同pH条件下黄单胞菌噬菌体GJ19P1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黄单胞菌噬菌体GJ19P1在不同pH条件下的稳定性
Figure PCTCN2021081420-appb-000005
结果如表4所示，在pH＝2～10之间，黄单胞菌噬菌体GJ19P1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＝2的酸性条件下，黄单胞菌噬菌体GJ19P1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，表明其在酸性条件下有较好的耐受性。
8-2：不同温度条件下黄单胞菌噬菌体GJ19P1的稳定性
将效价为1.0×10 7PFU/mL的黄单胞菌噬菌体GJ19P1(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黄单胞菌噬菌体GJ19P1在4℃条件下的稳定性
Figure PCTCN2021081420-appb-000006
表6地毯草黄单胞菌噬菌体GJ19P1在25℃条件下的稳定性
Figure PCTCN2021081420-appb-000007
Figure PCTCN2021081420-appb-000008
表7地毯草黄单胞菌噬菌体GJ19P1在40℃条件下的稳定性
Figure PCTCN2021081420-appb-000009
由表5～表7可知，4℃条件下，黄单胞菌噬菌体GJ19P1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，黄单胞菌噬菌体GJ19P1存放4周后效价无明显降低；40℃条件下，噬菌体GJ19P1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，黄单胞菌噬菌体GJ19P1在不同温度条件下都具有较好的稳定性。
实施例9 黄单胞菌噬菌体GJ19P1对紫外线的耐受能力测试
取10mL效价为1×10 8PFU/mL的地毯草黄单胞菌噬菌体GJ19P1(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黄单胞菌噬菌体GJ19P1在紫外线照射下的稳定性
Figure PCTCN2021081420-appb-000010
结果如表8所示，紫外线照射8h时，地毯草黄单胞菌噬菌体GJ19P1的效价下降2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 黄单胞菌噬菌体GJ19P1裂解谱实验
采用双层平板点滴法测定噬菌体的裂解谱。选取分属9种不同的黄单胞菌致病变种菌株，合计184株。分别挑取供试菌株单菌落，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GJ19P1溶液(由实施例7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黄单胞菌噬菌体GJ19P1裂解谱实验
Figure PCTCN2021081420-appb-000011
Figure PCTCN2021081420-appb-000012
Figure PCTCN2021081420-appb-000013
Figure PCTCN2021081420-appb-000014
Figure PCTCN2021081420-appb-000015
Figure PCTCN2021081420-appb-000016
Figure PCTCN2021081420-appb-000017
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GJ19P1对9种184株不同黄单胞菌致病变种菌株均具有较强的裂解能力，其裂解率可达91.8％；且其对上述单一黄单胞菌致病变种的裂解能力均在85％以上。噬菌体GJ19P1具有较宽的裂解谱。
实施例11 黄单胞菌噬菌体GJ19P1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3mL TSB液体培养基的试管中，37℃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GJ19P1溶液(由实施例7制得)滴于平板上，待自然风干后37℃培养12h，观察结果。
表10黄单胞菌噬菌体GJ19P1对非致病性有益细菌的裂解试验
Figure PCTCN2021081420-appb-000018
Figure PCTCN2021081420-appb-000019
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GJ19P1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 黄单胞菌噬菌体GJ19P1在液体中的杀菌效果
培养地毯草黄单胞菌GJ19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地毯草黄单胞菌GJ19的终浓度为5×10 3cfu/mL。向其中分别接入终浓度为2×10 2PFU/mL、2×10 3PFU/mL，2×10 4PFU/mL、2×10 5PFU/mL、2×10 6PFU/mL的黄单胞菌噬菌体GJ19P1(Xanthomonas phage GJ19P1)(实施例7制得)。同时设置对照组和空白组，对照组给予终浓度1x10 3cfu/mL的黄单胞菌噬菌体GJ19P1；空白组给予等量生理盐水。将各处理于28℃下150rpm振荡培养，4h后检测地毯草黄单胞菌的残留量。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。地毯草黄单胞菌数量＝TSA平板上菌落数量×稀释倍数×10。
表11不同浓度黄单胞菌噬菌体GJ19P1在液体中的杀菌效果
Figure PCTCN2021081420-appb-000020
Figure PCTCN2021081420-appb-000021
由表11可知，黄单胞菌噬菌体GJ19P1在其终浓度为1×10 2PFU/mL时，即可良好地控制地毯草黄单胞菌GJ19P1在液体培养基中的生长；当黄单胞菌噬菌体GJ19P1终浓度≥10 3PFU/mL时，其对地毯草黄单胞菌杀灭率可达97％以上。
实施例13 黄单胞菌噬菌体GJ19P1的组合物的制备
分别取效价为2x10 9PFU/mL黄单胞菌噬菌体GJ19P1(Xanthomonas phage GJ19P1)和地毯草黄单胞菌噬菌体YHC5(Xanthomonas axonopodis phage YHC5)的原液，将2株噬菌体等体积1:1均匀混合于SM液中，制成1:1的组合物(组合物1)。
分别取效价为2x10 9PFU/mL黄单胞菌噬菌体GJ19P1(Xanthomonas phage GJ19P1)和丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1(Pseudomonas syringae pv.Actinidiae phagePSA-P1)的原液，将2株噬菌体等体积均匀混合制成1:1的组合物(组合物2)。
分别取效价为2x10 9PFU/mL、黄单胞菌噬菌体GJ19P1(Xanthomonas phage GJ19P1)、茄科雷尔氏菌噬菌体GP3(Ralstonia solanacearum phageGP3)原液，将2株噬菌体等体积1:1均匀混合制成组合物(组合物3)。
分别取效价为2x10 9PFU/mL黄单胞菌噬菌体GJ19P1(Xanthomonas phage GJ19P1)、地毯草黄单胞菌噬菌体YHC5(Xanthomonas axonopodis phage YHC5)和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)的原液，将3株噬菌体等体积1:1:1均匀混合制成组合物(组合物4)。
分别取效价为2x10 9PFU/mL黄单胞菌噬菌体GJ19P1(Xanthomonas phage GJ19P1)、地毯草黄单胞菌噬菌体YHC5(Xanthomonas axonopodis phage YHC5)和茄科雷尔氏菌噬菌体GP3(Ralstonia solanacearum phage GP3)的原液，将3株噬菌体等体积1:1:1均匀混合制成组合物(组合物5)。
分别取效价为2x10 9PFU/mL黄单胞菌噬菌体GJ19P1(Xanthomonas phage GJ19P1)、丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1(Pseudomonas syringae pv.Actinidiae phagePSA-P1)和茄科雷尔氏菌噬菌体GP3(Ralstonia solanacearum phageGP3)的原液，将3株噬菌体等体积1:1:1均匀混合制成组合物(组合物6)。
分别取效价为2x10 9PFU/mL黄单胞菌噬菌体GJ19P1(Xanthomonas phage  GJ19P1)、地毯草黄单胞菌噬菌体YHC5(Xanthomonas axonopodis phage YHC5)、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)和茄科雷尔氏菌噬菌体GP3(Ralstonia solanacearum phage GP3)的原液，将4株噬菌体等体积1:1:1:1均匀混合制成组合物(组合物7)。
分别取效价为2x10 9PFU/mL黄单胞菌噬菌体GJ19P1(Xanthomonas phage GJ19P1)、终浓度50％的代森铵700倍液，将2者等体积1:1均匀混合制成组合物(组合物8)。
实施例14 黄单胞菌噬菌体GJ19P1组合物在液体中的杀菌效果
培养地毯草黄单胞菌至对数生长期，分装进不同试管中，用等体积的TSB液体培养基稀释菌液至地毯草黄单胞菌GJ19的终浓度为5×10 3cfu/mL，向其中分别接入实施例15制备的黄单胞菌噬菌体GJ19P1的组合物，使各组合物的终浓度为2×10 3PFU/mL，同时设置对照组和空白组，对照组给予终浓度5×10 3cfu/mL的地毯草黄单胞菌GJ19；空白组给予等量生理盐水。将各处理于28℃下150rpm振荡培养，4h后检测地毯草黄单胞菌GJ19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。地毯草黄单胞菌数量＝TSA平板上菌落数量×稀释倍数×10。
表12不同浓度地毯草黄单胞菌噬菌体GJ19P1组合物在液体中的杀菌效果
Figure PCTCN2021081420-appb-000022
由表12可知，黄单胞菌噬菌体GJ19P1的各个组合物对地毯草黄单胞菌均具有良好的杀灭作用。说明黄单胞菌噬菌体GJ19P1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。本实施例还可以与其它化学消毒剂联用，起到防杀效果。
实施例15 黄单胞菌噬菌体GJ19P1及其组合物对柑橘溃疡病的防治将终浓度分别为2×10 4PFU/mL、2×10 5PFU/mL、2×10 6PFU/mL的黄单胞菌噬菌体GJ19P1(Xanthomonas phage GJ19P1)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9、组合物10和组合物11。
柑橘树480棵，适应性培养1个月后，随机分为8组(噬菌体3组、组合物9～组合物11、对照组和空白组)，每组60棵，噬菌体实验组分别给予剂量为2×10 4PFU/mL、2×10 5PFU/mL、2×10 6PFU/mL的供试噬菌体(实施例7制得)和2×10 5cfu/mL的地毯草黄单胞菌GJ19；组合物实验组分别将各组合物给予2×10 5cfu/mL的地毯草黄单胞菌GJ19；对照组仅给予2×10 5cfu/mL的地毯草黄单胞菌GJ19；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内柑橘溃疡病的发病率，发病率＝发病株数/总株数×100％。
表13黄单胞菌噬菌体GJ19P1及其组合物对柑橘溃疡病的影响
Figure PCTCN2021081420-appb-000023
Figure PCTCN2021081420-appb-000024
由表13可知，对照组的柑橘树在接种15d时，柑橘溃疡病的发病率达到100％。而噬菌体各实验组中，黄单胞菌噬菌体GJ19P1浓度越高，柑橘树发病率越低；黄单胞菌噬菌体GJ19P1浓度为10 6PFU/mL时，攻毒15d后，柑橘树发病率保持在10％以内。表明黄单胞菌噬菌体GJ19P1及其组合物可以用作生物杀菌剂，有效防治柑橘溃疡病。
实施例16 黄单胞菌噬菌体GJ19P1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的黄单胞菌噬菌体GJ19P1液体或黄单胞菌噬菌体GJ19P1的组合物，1LTSB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黄单胞菌噬菌体GJ19P1液体或黄单胞菌噬菌体GJ19P1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黄单胞菌噬菌体GJ19P1液体或黄单胞菌噬菌体GJ19P1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17 黄单胞菌噬菌体GJ19P1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的黄单胞菌噬菌体GJ19P1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4主要成分为5～10mL的效价为3×10 8PFU/mL的茄科雷尔氏菌噬菌体GP3(Ralstonia solanacearum phage GP3)液体。
试剂盒5主要成分为5～10mL的效价为终浓度为50％的代森铵700倍液。
试剂盒6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GJ19P1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的茄科雷尔氏菌噬菌体GP3液体、5～10mL终浓度为50％的代森铵700倍液。
试验过程：柑橘树160棵，适应性培养1个月后，随机分为8组(试剂盒1组～试剂盒6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ACCC 03526(Xanthomonas axonopodis pv.citri)对试剂盒1组～试剂盒6组及阳性对照组共140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6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14黄单胞菌噬菌体GJ19P1及其组合物的试剂盒对柑橘溃疡病的影响
Figure PCTCN2021081420-appb-000025
Figure PCTCN2021081420-appb-000026
如表14，试剂盒6组的柑橘发病率较试剂盒1组的更低，说明含有所述组合物的试剂盒6对柑橘溃疡病防治显著，有所述组合物的试剂盒6较仅含有黄单胞菌噬菌体GJ19P1的试剂盒1在控制柑橘发病率方面的效果更佳。
实施例18 黄单胞菌噬菌体GJ19P1及其组合物的试剂盒对水稻细菌性条斑病的应用
试剂盒1～试剂盒6均采用实施例17的试剂盒。
试验过程：水稻植株160株，适应性培养1个月后，随机分为8组(试剂盒1～试剂盒6、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的稻黄单胞菌稻细条斑致病变种ACCC03523(Xanthomonas oryzaes pv.oryzicola)对试剂盒1～试剂盒6及阳性对照组共140株水稻植株进行攻毒处理，阴性对照组20株水稻植株分别以灌根法施入1L生理盐水。攻毒三天后，采用灌根法分别接种试剂盒1～试剂盒6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内水稻细菌性条斑病的病率，发病率＝发病株数/总株数×100％。
表15黄单胞菌噬菌体GJ19P1及其组合物的试剂盒对水稻细菌性条斑病的影响
Figure PCTCN2021081420-appb-000027
Figure PCTCN2021081420-appb-000028
如表15，试剂盒6组的水稻植株发病率较试剂盒1组的更低，说明含有所述组合物的试剂盒6对水稻细菌性条斑病防治显著，有所述组合物的试剂盒6较仅含有黄单胞菌噬菌体GJ19P1的试剂盒1在控制水稻植株发病率方面的效果更佳。
实施例19 黄单胞菌噬菌体GJ19P1及其组合物的试剂盒对木薯细菌性萎蔫病的应用
试剂盒1～试剂盒6均采用实施例17的试剂盒。
试验过程：木薯160株，适应性培养1个月后，随机分为8组(试剂盒1～试剂盒6、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的地毯草黄单胞菌木薯萎蔫致病变种ACCC 03517(Xanthomonas axonopodis pv.manihotis)对试剂盒1～试剂盒6及阳性对照组共140株木薯进行攻毒处理，阴性对照组20株木薯分别以灌根法施入1L生理盐水。攻毒三天后，采用灌根法分别接种试剂盒1～试剂盒6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内木薯细菌性萎蔫病的发病率，发病率＝发病株数/总株数×100％。
表16黄单胞菌噬菌体GJ19P1及其组合物的试剂盒对木薯细菌性萎蔫病的影响
Figure PCTCN2021081420-appb-000029
Figure PCTCN2021081420-appb-000030
如表16，试剂盒6组的木薯发病率较试剂盒1组的更低，说明含有所述组合物的试剂盒6对木薯细菌性萎蔫病防治显著，有所述组合物的试剂盒6较仅含有黄单胞菌噬菌体GJ19P1的试剂盒1在控制木薯发病率方面的效果更佳。
实施例20 黄单胞菌噬菌体GJ19P1及其组合物的试剂盒对芒果细菌性黑斑病的应用
试剂盒1～试剂盒6均采用实施例17的试剂盒。
试验过程：芒果树160棵，适应性培养1个月后，随机分为8组(试剂盒1～试剂盒6、阳性对照组和阴性对照组)，每组20株。采用茎干输液法分别将1L终浓度为1x10 3cfu/mL的野油菜黄单胞菌芒果致病变种ATCC 11637(Xanthomonas campestris pv.mangiferaeindicae)对试剂盒1～试剂盒6组及阳性对照组共140棵芒果树进行攻毒处理，阴性对照组20棵芒果树分别以茎干输液法施入1L生理盐水。攻毒三天后，采用茎干输液法分别接种试剂盒1～试剂盒6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内芒果细菌性黑斑病的发病率，发病率＝发病株数/总株数×100％。
表17黄单胞菌噬菌体GJ19P1及其组合物的试剂盒对芒果细菌性黑斑病的影响
Figure PCTCN2021081420-appb-000031
Figure PCTCN2021081420-appb-000032
如表17，试剂盒6组的芒果发病率较试剂盒1组的更低，说明含有所述组合物的试剂盒6对芒果细菌性黑斑病防治显著，有所述组合物的试剂盒6较仅含有黄单胞菌噬菌体GJ19P1的试剂盒1在控制发病率方面的效果更佳。
实施例21 黄单胞菌噬菌体GJ19P1及其组合物的试剂盒对猕猴桃溃疡病的应用
试剂盒1～试剂盒6均采用实施例17的试剂盒。
试验过程：猕猴桃树160棵，适应性培养1个月后，随机分为8组(试剂盒1～试剂盒6、阳性对照组和阴性对照组)，每组20株。采用茎干输液法分别将1L终浓度为1x10 3cfu/mL的丁香假单孢菌猕猴桃致病变种ATCC BAA-2502(Pseudomonassyringae pv.Actinidiae)对试剂盒1～试剂盒6及阳性对照组共140棵猕猴桃树进行攻毒处理，阴性对照组20棵猕猴桃树分别以茎干输液法施入1L生理盐水。攻毒三天后，采用注射法分别接种试剂盒1～试剂盒6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内猕猴桃溃疡病的发病率，发病率＝发病株数/总株数×100％。
表18黄单胞菌噬菌体GJ19P1及其组合物的试剂盒对猕猴桃溃疡病的影响
Figure PCTCN2021081420-appb-000033
如表18，试剂盒6组的猕猴桃发病率较试剂盒1组的更低，说明含有所述组合物的试剂盒6对猕猴桃溃疡病防治显著，有所述组合物的试剂盒6较仅含有黄单胞菌噬菌体GJ19P1的试剂盒1在控制发病率方面的效果更佳。
实施例22 黄单胞菌噬菌体GJ19P1及其组合物的试剂盒对生姜姜瘟病的应用
试剂盒1～试剂盒6均采用实施例17的试剂盒。
试验过程：生姜160株，适应性培养1个月后，随机分为8组(试剂 盒1～试剂盒6、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的茄科雷尔氏菌ATCC11696D-5(Ralstonia solanacearum)对试剂盒1～试剂盒6组及阳性对照组共140株生姜进行攻毒处理，阴性对照组20株生姜分别以注射法施入1L生理盐水。攻毒三天后，采用注射法分别接种试剂盒1～试剂盒6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内生姜姜瘟病的发病率，发病率＝发病株数/总株数×100％。
表19黄单胞菌噬菌体GJ19P1及其组合物的试剂盒对生姜姜瘟病的影响
Figure PCTCN2021081420-appb-000034
如表19，试剂盒6组的生姜发病率较试剂盒1组的更低，说明含有所述组合物的试剂盒6对生姜姜瘟病防治显著，有所述组合物的试剂盒6较仅含有黄单胞菌噬菌体GJ19P1的试剂盒1在控制发病率方面的效果更佳。
综上，本申请的黄单胞菌噬菌体GJ19P1(Xanthomonas phage GJ19P1),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，防治但不限于由黄单胞菌引起的各类细菌性疾病。
通过实施例17～22，可以看出，单独使用黄单胞菌噬菌体GJ19P1对攻毒实验所用的菌均具有杀灭效果，可对攻毒植株的病害起显著防治作用，但含有黄单胞菌噬菌体GJ19P1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 (10)

  1. 一种可跨种裂解的黄单胞菌噬菌体，其特征在于，所述黄单胞菌噬菌体为黄单胞菌噬菌体GJ19P1(Xanthomonas phage GJ19P1)，保藏编号为CCTCC NO：M 2020805。
  2. 根据权利要求1所述的一种可跨种裂解的黄单胞菌噬菌体，其特征在于：所述黄单胞菌噬菌体GJ19P1(Xanthomonas phage GJ19P1)在感染复数MOI＝0.0001条件下培养24h，效价达到3.5×10 10PFU/mL以上。
  3. 根据权利要求1所述的一种可跨种裂解的黄单胞菌噬菌体，其特征在于：所述黄单胞菌噬菌体GJ19P1(Xanthomonas phage GJ19P1)在pH＝2～10的条件下具有耐性，在96h内效价降低不超过4个数量级；黄单胞菌噬菌体GJ19P1(Xanthomonas phage GJ19P1)对9种不同黄单胞菌致病变种菌株均具有较强的裂解能力，裂解率可达91.8％；且黄单胞菌噬菌体GJ19P1(Xanthomonas phage GJ19P1)对单一黄单胞菌致病变种的裂解能力均在85％以上。
  4. 根据权利要求1所述的一种可跨种裂解的黄单胞菌噬菌体，其特征在于：所述黄单胞菌噬菌体GJ19P1(Xanthomonas phage GJ19P1)经紫外辐射8h后，效价降低不超过2个数量级。
  5. 含有权利要求1～4任意一项所述的一种可跨种裂解的黄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黄单胞菌噬菌体GJ19P1(Xanthomonas phage GJ19P1)。
  6. 根据权利要求5所述的一种可跨种裂解的黄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。
  7. 根据权利要求6所述的一种可跨种裂解的黄单胞菌噬菌体的组合物，其特征在于：所述化学性杀菌剂为代森铵700倍液。
  8. 根据权利要求5所述的一种可跨种裂解的黄单胞菌噬菌体的组合物，其特征在于：所述组合物包括地毯草黄单胞菌噬菌体YHC5(Xanthomonas axonopodis phage YHC5)，保藏编号为CCTCC NO:M 2018579；丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1(Pseudomonas syringae pv.Actinidiae phagePSA-P1)，保藏编号为CCTCC NO:M 2020252；茄科雷尔氏菌噬菌体GP3(Ralstonia solanacearum phageGP3)，保藏编号为CCTCC NO:M2016635。
  9. 含有权利要求8所述的一种可跨种裂解的黄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黄单胞菌噬菌体GJ19P1(Xanthomonas phage GJ19P1)或黄单胞菌噬菌体GJ19P1(Xanthomonas phage GJ19P1)的组合物。
  10. 一种可跨种裂解的黄单胞菌噬菌体组合物的应用，其特征在于：权利要求6～8任意一项所述的黄单胞菌噬菌体GJ19P1的组合物，用作为生物类消毒剂或生物类农药的有效成分，防治但不限于由黄单胞菌的细菌性疾病。
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