CN113151192B - Xanthomonas phage capable of cross-species lysis, composition, kit and application thereof - Google Patents
Xanthomonas phage capable of cross-species lysis, composition, kit and application thereof Download PDFInfo
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- CN113151192B CN113151192B CN202110243645.8A CN202110243645A CN113151192B CN 113151192 B CN113151192 B CN 113151192B CN 202110243645 A CN202110243645 A CN 202110243645A CN 113151192 B CN113151192 B CN 113151192B
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Abstract
The invention relates to the field of microorganisms, and particularly discloses a xanthomonas bacteriophage capable of cross-species lysis, a composition, a kit and application thereof, wherein the xanthomonas bacteriophage is xanthomonas bacteriophage GJ19P 1%Xanthomonasphage GJ19P 1) with a preservation number of CCTCC NO: M2020805; the phage has higher tolerance to ultraviolet and acid environments, and can play a better role in preventing and controlling in different environments; the composition at least comprises a xanthomonas phage GJ19P1; a composition with Xanthomonas phage GJ19P1 or Xanthomonas phage GJ19P1 in the kit; the composition of Xanthomonas phage GJ19P1 is useful for, but not limited to, killing Xanthomonas.
Description
Technical Field
The invention relates to the field of phage, in particular to a cross-species cleavable Xanthomonas phage, and a composition, a kit and application thereof.
Background
Xanthomonas, straight rod-like bacteria, terminal flagellum movement, obligate aerobics. A water insoluble yellow pigment, a carotenoid, is produced on the medium and its chemical composition is bromoarylpolyene, which makes the colony yellow. All xanthomonas are plant pathogenic bacteria that can cause plant diseases.
A total of 14 pathogenic varieties in Xanthomonas are listed in the name of plant quarantine harmful organisms in the people's republic of China because of important quarantine significance, and specifically comprise: the species may be selected from the group consisting of Saccharum sinensis (Xanthomonas albilineans), rhizoctonia necatrix (Xanthomonas arboricola pv. Celebensis), rhizoctonia cerealis pepper pathogenic variant (Xanthomonas axonopodis pv. betlicola), rhizoctonia cerealis pathogenic variant (Xanthomonas axonopodis pv. Citri), rhizoctonia cerealis cassava wilt pathogenic variant (Xanthomonas axonopodis pv. Mannihotis), rhizoctonia cerealis sugarcane pathogenic variant (Xanthomonas axonopodis pv. Vasculorium), rhizoctonia cerealis pathogenic variant (Xanthomonas axonopodis pv. Mangifer eindiae), xanthomonas campestris mango pathogenic variant (Xanthomonas campestris pv. Mangiferaeeindiae), xanthomonas campestris pathogenic variant (Xanthomonas campestris pv. Musaceae), rhizoctonia cerealis (Xanthomonas cassavae), strawberry angular leaf spot (Xanthomonas fragariae), rhizoctonia solani (Xanthomonas hyacinthi), rhizoctonia oryzae white leaf blight pathogenic bacterium (Xanthomonas oryzae v. Oryza), rhizoctonia cerealis pathogenic variant (Xanthomonas oryzae pv. Zii and Rhizoctonia solani (sora) and Rhizoctonia solani (Xanthomonas populi). According to the statistical display of port interception data of animal and plant quarantine information resource sharing service platform of China inspection and quarantine science institute, each port branch office in China intercepts xanthomonas amounting 586 batch in passenger carrying object and cargo quarantine from 2015 1 month to 2017 2 months, and related cargoes are mainly grain, soybean, vegetables, fruits, other plant products, seedlings, raw materials and products, logs and the like. For example, bacterial leaf blight caused by xanthomonas oryzae is one of the bacterial diseases that are very serious in rice production. Mango bacterial black spot caused by xanthomonas campestris mango pathogenic variety is a frequently-occurring important disease on mango, and the disease seriously affects mango yield and commodity value of fruits. The pathogenic varieties of xanthomonas carpet are pathogenic bacteria causing citrus canker, and seriously affect the yield and quality of citrus fruits. (Lin Wenli, sho Fu Lian. Method for detecting citrus canker and progress of control technique [ J ]. Modern agricultural science and technology 2012 (22): 112-114.).
Phages are a kind of viruses which are specially used for cracking bacteria, and the main chemical components of the phages are composed of proteins and nucleic acids, are widely used in soil, air, water and organisms, have stronger specificity, and kill viruses of specific bacteria after being combined with specific sites on the surfaces of bacterial cells. Since Frederik Tword first discovered phages in 1915, more and more studies have demonstrated that phages have higher antimicrobial activity and specificity, and can control drug-resistant bacteria and prevent damage to microbial communities. Compared with antibiotic treatment, the side effect is extremely low and more rapid and effective, does not inhibit the natural immunity of the organism or cause allergic reaction, and has no infectivity and toxicity to human beings, other mammals and plants. However, the current research on phage is focused on screening and biological property discussion, and has been recently reported on application. Therefore, screening and application of suitable virulent phages is an effective way to develop novel antibacterial agents.
Currently, there is little research associated with the use of Xanthomonas phages for controlling plant bacterial diseases caused by Xanthomonas.
In view of the above related art, the inventors considered that there is an urgent need to find a xanthomonas phage that can be cross-species lysed.
Disclosure of Invention
In order to solve the technical problem of tolerance of phage in different control environments, the application provides a xanthomonas phage capable of cross-species lysis, a composition, a kit and application thereof.
In a first aspect, the application provides a cross-species cleavable Xanthomonas phage, which adopts the following technical scheme:
a cross-species cleavable Xanthomonas phage is Xanthomonas phage GJ19P1 (Xanthomonas phage GJ19P 1), and the preservation number is CCTCC NO: M2020805.
By adopting the technical scheme, the application provides a Xanthomonas phage GJ19P1 which has excellent pH resistance, and is stored in China center for type culture collection with a collection number of CCTCC NO: M2020805.
Preferably, the Xanthomonas phage GJ19P1 (Xanthomonas phage GJ19P 1) is cultured under the condition that the multiplicity of infection MOI=0.0001 for 24 hours, and the titer reaches 3.5X10 10 PFU/mL or more.
By adopting the technical scheme, the multiplicity of infection (MOI) is the ratio of the number of phage to the number of bacteria, and is an important basis for researching the quantitative effect relationship between phage infected bacteria and produced phage filial generation. The Xanthomonas phage can infect Xanthomonas and proliferate to obtain a large number of sub-phage. The application provides a high-quality phage strain source for industrial production of phage bactericides.
Preferably, the xanthomonas phage GJ19P1 (Xanthomonas phage GJ19P 1) is resistant at ph=2-10, with a potency decrease of no more than 4 orders of magnitude within 96 hours; the xanthomonas phage GJ19P1 (Xanthomonas phage GJ19P 1) has strong cracking capability on 9 different xanthomonas pathogenic strain strains, and the cracking rate can reach 91.8%; and the splitting ability of the Xanthomonas phage GJ19P1 (Xanthomonas phage GJ P1) to single Xanthomonas pathogenic variant is above 85 percent
By adopting the technical scheme, the Xanthomonas phage GJ19P1 has excellent tolerance under the condition of pH=2, namely under the acidic condition, and can play a good role in preventing and controlling when the preventing and controlling environment is between pH=2 and 10. The Xanthomonas phage GJ19P1 of the present embodiment can be used to control bacterial diseases caused by a variety of Xanthomonas pathogenic varieties.
Preferably, the titer of the Xanthomonas phage GJ19P1 (Xanthomonas phage GJ19P 1) is reduced by no more than 2 orders of magnitude after 8 hours of ultraviolet radiation.
By adopting the technical scheme, when ultraviolet rays in the prevention and treatment environment are strong, the Xanthomonas phage GJ19P1 has good tolerance, less titer reduction and can play a good prevention and treatment role on pathogenic bacteria.
Preferably, the Xanthomonas phage GJ19P1 (Xanthomonas phage GJ19P 1) can efficiently identify different Xanthomonas pathogenic varieties, and has a wider lysis range.
By adopting the technical scheme, the xanthomonas phage GJ19P1 has strong cracking capacity on 9 different xanthomonas pathogenic variant strains, and the cracking rate can reach 91.8%; and the cracking ability of the strain to single pathogenic variant of Xanthomonas is above 85%. The xanthomonas phage GJ19P1 can be used for preventing and controlling bacterial diseases caused by various xanthomonas pathogenic varieties.
In a second aspect, the application provides a cross-species cleavable Xanthomonas phage composition, which adopts the following technical scheme:
a composition of cross-species cleavable xanthomonas phage, said composition comprising at least one xanthomonas phage GJ19P1 (Xanthomonas phage GJ19P 1).
Preferably, the composition comprises a chemical biocide.
Preferably, the chemical bactericide is amikacin 700 times liquid.
By adopting the technical scheme, the Xanthomonas phage GJ19P1 and the chemical bactericide are used as a composition in a combined way. As an exemplary illustration, the proportional relationship between xanthomonas phage GJ19P1 and the 700-fold solution of ambam can be determined by one skilled in the art in combination with the present application and the actual field of application and general knowledge in the art.
Preferably, the composition comprises xanthomonas carpet grass phage YHC5 (Xanthomonas axonopodis phage YHC), and the preservation number is CCTCC NO: M2018579; pseudomonas syringae kiwi fruit pathogenic variant phage PSA-P1 (Pseudomonas syringae pv. Actinidiae phagePSA-P1) with a preservation number of CCTCC NO: M2020252; the Solanaceae Ralstonia phage GP3 (Ralstonia solanacearum phageGP) has a preservation number of CCTCC NO: M2016635.
By adopting the technical scheme, the Xanthomonas phage GJ19P1 (Xanthomonas phage GJ19P 1) can be mixed with other phages for use, and the composition has a cracking rate of over 97 percent on Xanthomonas carpet and has stronger cracking property.
In a third aspect, the invention provides a kit for cross-species lytic xanthomonas phage, which adopts the following technical scheme:
a kit of cross-species cleavable xanthomonas phage with a composition of said xanthomonas phage GJ19P1 (Xanthomonas phage GJ19P 1) or xanthomonas phage GJ19P1 (Xanthomonas phage GJ P1).
Through the technical scheme, the Xanthomonas phage provided by the invention is applied to rapid detection of Xanthomonas, and comprises the steps of detecting Xanthomonas in a test paper, a test paper box and the like, or screening target pathogenic bacteria in a clinical sample, so that the detection sensitivity is effectively ensured.
In a fourth aspect, the invention provides an application of a cross-species cleavable Xanthomonas phage composition, which adopts the following technical scheme:
the application of a cross-species cleavable Xanthomonas phage composition, wherein the Xanthomonas phage GJ19P1 composition is used as an active ingredient of biological disinfectants or biological pesticides for preventing and treating but not limited to bacterial diseases caused by Xanthomonas.
By adopting the technical scheme, the Xanthomonas phage GJ19P1 and the composition thereof can be used for treating and preventing bacterial infection caused by Xanthomonas and are not limited to Xanthomonas and can be used as a biological agent for preventing and treating diseases caused by Xanthomonas and are not limited to Xanthomonas.
In summary, the invention has the following beneficial effects:
1. the xanthomonas phage GJ19P1 has higher tolerance to ultraviolet rays and acidic environments, is suitable for different control environments, and can have better biological control effect on citrus canker;
2. the xanthomonas phage GJ19P1 is a virulent phage separated from nature, the tested phage does not contain virulence genes or bad genes, the DNA of the phage cannot encode proteins which can cause potential health risks, and the possibility of carrying lysogenic genes does not exist;
3. The xanthomonas phage GJ19P1 has high affinity and cleavage capacity, and can reach 10 in 24 hours of culture 10 Titers above PFU/mL; xanthomonas phage GJ19P1 can specifically partially or completely inactivate Xanthomonas, can complete mass proliferation with a small amount of initial phage, and provides a high-quality phage strain source for industrial phage bactericides; the xanthomonas phage GJ19P1 or the composition thereof can be prepared into various products applied to detection, disinfection, plant protection and other aspects by a person skilled in the art according to the description of the application and common general knowledge in the art, and is industrially applied;
4. the Xanthomonas phage GJ19P1 is a strict virulent phage, has high specificity and cracking property for host bacteria, has a wider host range, and has a cracking rate of 91.8% for 9 different Xanthomonas pathogenic varieties of 184 strains; and the cracking ability to single Xanthomonas pathogenic variety is above 85%; xanthomonas phage GJ19P1 can be used as an active ingredient of various products applied to environmental disinfection, for example, including but not 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 soaking, spraying, combined use with aqueous carriers, and the like, and can effectively control the growth and activity of target bacteria; such liquid immersion, spray forms include, but are not limited to, detergents, disinfectants, decontaminating agents, and the like; such aqueous carriers include, but are not limited to, phosphate buffer, TSB medium, LB medium, chlorine free water, and the like;
5. The Xanthomonas phage GJ19P1 of the application can not identify any one of 10 strains of tested non-host pathogenic bacteria, which shows that the specificity is good;
6. the Xanthomonas phage GJ19P1 has good stability, has tolerance under the condition of pH=2-10, and reduces the titer by not more than 4 orders of magnitude within 96 hours; after 8 hours of ultraviolet radiation, the potency is reduced by not more than 2 orders of magnitude;
7. the Xanthomonas phage GJ19P1 can be used for preparing a composition, a reagent or a kit, is applied to the rapid detection of Xanthomonas, and comprises the steps of detecting Xanthomonas in a target sample in a test paper, a kit and the like, or screening target pathogenic bacteria in a clinical sample, so that the detection sensitivity is effectively ensured;
8. xanthomonas phage GJ19P1 and composition thereof at a concentration of 10% in liquid medium 3 The Xanthomonas campestris of PFU/mL has good killing capacity; when the concentration of the xanthomonas phage GJ19P1 is more than or equal to 10 4 When PFU/mL, the killing rate of the medicine to Xanthomonas is more than 98.2%, and the medicine has no antagonism to other combined substances;
9. the xanthomonas phage GJ19P1 (Xanthomonas phageGJ P1) and the composition thereof of the present application can be prepared by a person skilled in the art according to the description of the present application and general knowledge in the art into a biopharmaceutical applicable to control of diseases caused by xanthomonas, and not limited to xanthomonas.
Drawings
FIG. 1 is a schematic of plaques of Xanthomonas phage GJ19P1 (Xanthomonas phageGJ P1).
FIG. 2 is a schematic diagram of morphological structure of the X.Xanthomonas phage GJ19P1 (Xanthomonas phage GJ19P 1) identified by electron microscopy.
Detailed Description
The invention is described in further detail below with reference to fig. 1 and the examples.
In the following examples, the strain numbers referred to are all named numbers of the company.
Xanthomonas phage GJ19P1 (Xanthomonas phage GJ P1) with a preservation number of CCTCC NO: M2020805, a preservation unit of China center for type culture Collection, a preservation time of 11 months and 30 days in 2020, and a preservation unit address of university of Wuhan, china;
xanthomonas carpet grass phage YHC5 (Xanthomonas axonopodis phage YHC) with a preservation number of CCTCC NO: M2018579, a preservation unit of China center for type culture Collection, a preservation time of 2018, 08 and 30 days, and a preservation unit address of university of Chinese, wuhan.
Pseudomonas syringae kiwi fruit pathogenic variant phage PSA-P1 (Pseudomonas syringae pv. Actinidiae phagePSA-P1) with a preservation number of CCTCC NO: m2020252, the preservation unit is China center for type culture Collection, the preservation time is 2020, 06 and 30 days, and the preservation unit address is China university of Wuhan.
The preservation unit of the solanaceae Ralstonia phage GP3 (Ralstonia solanacearum phage GP 3) is China center for type culture collection, and the address is Chinese university of Wuhan, mail code 430072; the preservation date is 2016, 11 and 10; the preservation number is CCTCC NO: m2016635.
Xanthomonas carpet citrus pathogenic variant (Xanthomonas axonopodis pv. Citri), deposit number ACCC 03526, was purchased in connection with the depository.
Xanthomonas carpet, cassava wilt disease variants (Xanthomonas axonopodis pv. manihotis), deposit number ACCC 03517, are available by contact purchase from the depository.
Xanthomonas campestris mango pathogenic variant (Xanthomonas campestris pv. mangifetaeindicae), deposit number ATCC 11637, is commercially available from contact with the depositary.
Xanthomonas oryzae Oryza sativa pathogenic variety (Xanthomonas oryzae pv. Oryzicola) with accession number ACCC03523, was purchased in connection with the depository.
Pseudomonas syringae, actinidiae, deposit number ATCC BAA-2502, is available from a commercial connection to a depository.
Ralstonia solanacearum (Ralstonia solanacearum), deposit number ATCC11696D-5, is available from contact with the depository.
In the examples to be given below,
the formula of the TSB liquid culture medium is as follows: 15g of tryptone, 5g of soybean peptone, 5g of sodium chloride and 1000mL of distilled water;
the formula of the TSA solid culture medium is as follows: 15g of tryptone, 5g of soybean peptone, 5g of sodium chloride, 15g of agar and 1000mL of distilled water;
TSA plates: sterilizing the TSA solid culture medium, pouring the TSA solid culture medium onto a sterile plate, and cooling and solidifying the TSA solid culture medium to prepare a TSA plate;
the formula of the TSB semi-solid agar medium is as follows: 15g of tryptone, 5g of soybean peptone, 5g of sodium chloride, 7g of agar and 1000mL of distilled water;
SM liquid formula: 5.8g of sodium chloride, 2g of magnesium sulfate, 50mL of 1mol/L Tris-HCl, 0.25g of gelatin and 1000mL of distilled water.
Example 1 isolation preparation and purification culture of Xanthomonas phage GJ19P 1A source sample of Xanthomonas phage GJ19P1 in the present invention was collected from sewage in Jiang Ning region of Nanjing, jiangsu province, filtered with double-layer filter paper, centrifuged at low speed and at normal temperature, and the supernatant was filtered with 0.22 μm filter film.
Isolation of phages:
(1) Taking 10mL of filtered supernatant, adding the 10mL of filtered supernatant into 10mL of 2-time TSB liquid culture medium, simultaneously adding 1mL of phage host bacterium GJ19 logarithmic phase bacterial liquid, and placing the mixture at 28 ℃ for overnight culture;
(2) Centrifuging the culture at 8000rpm for 10min, and filtering supernatant with 0.22 μm filter membrane;
(3) Taking 0.5mL of phage host bacterium Xanthomonas carpet grass GJ19 log phase bacterial liquid, adding into 5mL of TSB semi-solid agar medium at 40 ℃ for uniform mixing, pouring onto a TSA plate, and preparing a double-layer plate containing host bacteria;
(4) 10 mu L of the supernatant is taken and dripped on a coagulated double-layer plate, and after air-dried under a sterile condition, the supernatant is placed at 28 ℃ for overnight culture to form phage spot.
Purification of phage:
(1) Picking phage spot by toothpick, transferring to 1mL SM solution, and shaking at room temperature for 1min;
(2) Performing 10-fold gradient dilution to obtain 10 2 、10 4 And 10 6 Respectively adding 0.5mL of phage host bacteria log phase bacterial liquid into the diluted liquid, and uniformly mixing;
(3) Standing for 15min, adding the mixed solution into 5mL of TSB semi-solid agar medium at 45 ℃, uniformly mixing, immediately pouring the mixed solution onto a TSA flat plate, shaking and flattening for 5min, standing for solidification, standing in a 28 ℃ incubator overnight for culture, and observing to obtain a double-layer flat plate containing single plaque;
(4) Picking up single plaque, transferring to 1mL SM solution, purifying for at least 3 times according to the above method, and finally forming plaque with consistent shape and size on a flat plate;
(5) Picking single plaque with consistent shape and size by toothpick, placing in 50mL TSB liquid culture medium containing 1mL logarithmic phase host bacterial liquid, shaking culture at 28deg.C at 180rpm overnight;
(6) Centrifuging the culture at 8000rpm for 10min, and filtering the supernatant with 0.22 μm filter membrane to obtain purified phage solution, namely Xanthomonas phage GJ19P1 (Xanthomonas phage GJ19P 1). Xanthomonas phage GJ19P1 (Xanthomonas phage GJ P1) produced a single, center-transparent, circular plaque of 6-7 mm diameter on Xanthomonas carpet, see FIG. 1. Xanthomonas phage GJ19P1 (Xanthomonas phage GJ P1) with a preservation number of CCTCC M:2020805.
Example 2 Electron microscopic observations of Xanthomonas phage GJ19P1
The purified phage solution prepared in example 1 was taken and observed by electron microscopy: and (3) dripping 20 mu L of sample on the copper mesh, naturally precipitating for 15min, absorbing excessive liquid from the side by using filter paper, adding 1 drop of 2% phosphotungstic acid on the copper mesh, dyeing for 10min, absorbing dye liquor from the side by using filter paper, drying and observing by using an electron microscope.
As a result, as shown in FIG. 2, the morphology of Xanthomonas phage GJ19P1 was observed under an electron microscope, and the phage exhibited 1 polyhedral stereosymmetrical head with a diameter of about 60nm under an electron microscope, and a short contracted tail. The phage belongs to the family of self-replicating short-tailed phages (Autographiviridae).
Example 3 preparation of Xanthomonas phage GJ19P1 particles and extraction and sequencing of genome
(1) Taking 100mL of the purified phage solution prepared in the example 1, sequentially adding DNaseI 20 mu L, RNaseA mu L with the concentration of 5mg/mL, incubating at 37 ℃ for 60min, adding 5.84g NaCl, and placing in an ice bath for 1h after dissolution;
(2) Centrifuging at 11000rpm for 10min at 4deg.C, transferring the supernatant to a new centrifuge tube, adding solid PEG8000 to obtain final concentration of 10% (w/v), and ice-bathing for 1 hr after PEG8000 is completely dissolved;
(3) Centrifuging at 11000rpm for 20min at 4deg.C, adding 1mLSM solution, and re-suspending to obtain concentrated phage particle solution, and preserving at 4deg.C.
Phage nucleic acids were extracted and sequenced using lambda phage genomic DNA kit. Through nucleotide sequencing, the Xanthomonas phage GJ19P1 (Xanthomonas phage PSA-P1) has the nucleotide sequence shown in SEQ ID No. 1.
Example 4 determination of the titer of Xanthomonas phage GJ19P1
Stock solution of Xanthomonas phage GJ19P1 (prepared in example 1) was serially diluted to l0 in a 10-fold gradient using SM solution as a diluent 8 Multiple times. Respectively taking l0 5 、l0 6 、l0 7 L0 8 The diluted phage culture liquid L000 mu L and the host bacterial liquid 300 mu L are uniformly mixed, and the mixture is kept stand for 15min to enable the phage culture liquid L000 mu L and the host bacterial liquid to be fully combined with the receptor on the bacterial surface. Adding the mixed solution into 4mL of semisolid agar culture medium cooled to 50 ℃, uniformly mixing, immediately spreading on a solidified solid agar plate, and inversely culturing for 6-8 h at 28 ℃ after agar solidification. Three replicates were made for each dilution and the average of the three replicates for this dilution was taken at the time of counting. Wherein phage titer (PFU/mL) =average plaque number x dilution fold
As can be seen from Table 1, xanthomonas phage GJ19P1 had 10 after 12h of culture 10 Titers above PFU/mL.
TABLE 1 titers of Xanthomonas phage GJ19P1 after 12h of culture
| Cultivation time | 4h | 8h | 12h |
| Phage GJ19P1 titer (PFU/mL) | 5.2x10 9 | 2.8x10 10 | 4.6x10 10 |
Example 5 test for detecting deletion of virulence Gene or adverse Gene of Xanthomonas phage GJ19P1
In this example 103 virulence genes identified as originating from lysogenic phages in pathogenic bacteria were selected as shown in Table 2, and the whole genome of Xanthomonas phage GJ19P1 was determined and subjected to bioinformatic analysis to determine whether it contained the following virulence genes. The results show that the Xanthomonas phage GJ19P1 does not contain the following virulence genes or adverse genes, so that it cannot encode proteins that may pose a potential health risk, and therefore the Xanthomonas phage GJ19P1 does not affect the health of the human or animal body.
TABLE 2 major known virulence genes of lysogenic phages in pathogenic bacteria
Example 6 toxicity experiment
The experimental mice were divided into two groups (phage group, control group) at random, 10 groups (5 animals and females) each, and the phage group was given a dose of 10 after three days of adaptive breeding of 20 animals and females each 10 PFU/kg Xanthomonas phage GJ19P1, control group was given equal amount of physiological saline, and the mice were sacrificed after neck-breaking and examined for visceral condition by continuous administration for 15 d.
Experimental results show that the dose of Xanthomonas phage GJ19P1 has no effect on daily mouse behavior. No abnormality was seen in the dissected examination viscera. Xanthomonas phage GJ19P1 has biological safety and can be used as a crop disease control preparation.
Example 7 determination of the optimal multiplicity of infection (MOI) of Xanthomonas campestris by Xanthomonas phage GJ19P1
A single colony of the host strain Xanthomonas carpet GJ19 is selected, inoculated into a test tube containing 3mL of TSB liquid culture medium, and subjected to overnight shaking culture in a shaking table at the temperature of 28 ℃ at 180rpm to obtain a host strain suspension. The host bacterial suspension was transferred to 10mL of TSB liquid medium at a ratio of 1:100, and cultured with shaking at 28℃and 180rpm until the logarithmic phase. Phage GJ19P1 purified solution (prepared in example 1) and phage host bacteria (MOI = purified phage solution titer/phage host bacteria concentration) were added in proportions of 100, 10, 1, 0.1, 0.01, 0.001 and 0.0001, 0.00001, 0.000001, 0.0000001, respectively, and the total volumes of the tubes were made the same by adding TSB liquid medium. The culture was carried out with shaking at 160rpm in a shaker at 28℃for 12h. 10000g of the culture medium is centrifuged for 10min after the culture is finished, and the supernatant culture solution is collected, and the titer of each processed phage is determined by adopting a double-layer plate method. Each point was averaged in duplicate replicates to produce the highest phage titer MOI as the optimal multiplicity of infection. Experiments were repeated 3 times.
TABLE 3 titers of Xanthomonas phage GJ19P1 at different infectious complex numbers
| MOI | GJ19P1(PFU/mL) | Host bacterium (cfu/mL) | GJ19P1 potency (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 |
As is clear from Table 3, the phage GJ19P1 titer reached the maximum of 3.5X10 under the condition of 12h of culture 10 PFU/mL, moi=0.0001. It shows that only a small amount of initial Xanthomonas phage GJ19P1 is needed to complete the mass proliferation. Xanthomonas phage GJ19P1 provides a source of high-quality phage strains for the industrial production of phage bactericides.
Example 8 determination of the pH and temperature stability of Xanthomonas phage GJ19P1
8-1: stability of Xanthomonas phage GJ19P1 at different pH conditions
Taking sterile EP tube, adding 900 μl of TSB liquid culture medium with pH=1-14, placing the EP tube in a constant temperature water bath at 25deg.C, adding L00 μl purified phage solution (prepared in example 7) after temperature balancing, and making initial titer of 1×10 10 PFU/mL, left to stand at room temperature. Samples were taken at 1h, 4h, 8h, 24h and 96h, respectively, and phage titers were determined by double-layer plate method after appropriate dilution of each treated sample. Experiments were repeated 3 times.
TABLE 4 stability of Xanthomonas phage GJ19P1 at different pH conditions
The results are shown in table 4, and the titer of the xanthomonas phage GJ19P1 is not changed significantly between ph=2 and 10, indicating that the xanthomonas phage GJ19P1 has better stability under neutral, slightly acidic and slightly alkaline conditions.
The titer of xanthomonas phage GJ19P1 was somewhat reduced at ph=2, but about 3 orders of magnitude lower than at ph=7, indicating better tolerability under acidic conditions.
8-2: stability of Xanthomonas phage GJ19P1 at different temperature conditions will have a titer of 1.0X10 7 PFU/mL Xanthomonas phage GJ19P1 (prepared in example 7) was subjected to periodic sampling at 4 ℃,25℃and 40℃respectively, and the titers were determined.
TABLE 5 stability of Xanthomonas phage GJ19P1 at 4℃
TABLE 6 stability of Xanthomonas carpet phage GJ19P1 at 25℃
TABLE 7 stability of Xanthomonas carpet phage GJ19P1 at 40℃
As can be seen from tables 5 to 7, the Xanthomonas phage GJ19P1 has good stability at 4 ℃, the titer is not obviously reduced after the Xanthomonas phage GJ19P1 is stored for 3 months, and the titer is still not reduced by more than 1 order of magnitude after the Xanthomonas phage GJ19P1 is stored for 12 months; the titer of the Xanthomonas phage GJ19P1 is not obviously reduced after the Xanthomonas phage GJ19P1 is stored for 4 weeks at the temperature of 25 ℃; at 40 ℃, the titer of phage GJ19P1 was not significantly reduced within 24 hours, and after 72 hours, the titer was reduced by 1 order of magnitude. Therefore, the Xanthomonas phage GJ19P1 has better stability under different temperature conditions.
Example 9 resistance test of Xanthomonas phage GJ19P1 to ultraviolet rays 10mL of the titer was taken as 1X 10 8 PFU/mL carpet grass xanthomonas phage GJ19P1 (prepared in example 7) was spread in a 90mm sterile petri dish and placed in an ultra clean bench and irradiated under an ultraviolet lamp (20 w, 20 cm). Samples were taken at 0min, 20min, 40min, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, respectively, and the phage titers were determined by a double-layer plate method after placing them in the dark for 30 min.
TABLE 8 stability of Xanthomonas phage GJ19P1 under ultraviolet irradiation
As a result, as shown in Table 8, the titer of Xanthomonas carpet phage GJ19P1 was reduced by 2 orders of magnitude when irradiated with ultraviolet rays for 8 hours, and therefore, the phage of the present invention was highly resistant to ultraviolet rays.
EXAMPLE 10 Xanthomonas phage GJ19P1 cleavage Spectrum experiment
The lytic spectrum of phage was determined by double-layer plate spot method. The total 184 strains of the pathogenic varieties of xanthomonas which belong to 9 different varieties are selected. The single colony of the strain to be tested is selected and inoculated into a test tube containing 3mL TSB liquid culture medium, and the strain is cultured overnight at 28 ℃ at 180rpm, so that bacterial solutions of each strain are prepared. mu.L of the bacterial suspension was mixed with TSB semi-solid agar medium and spread on a common agar plate, 5. Mu.L of purified phage GJ19P1 solution (prepared in example 7) was dropped on the plate, and after natural air-drying, the culture was carried out at 28℃overnight, and the results were observed.
TABLE 9 Xanthomonas phage GJ19P1 cleavage Spectrum experiment
Note that: "+". ++'s completely transparent; "++" medium clear; "+" is slightly clear; non-cleavable is "-".
As shown in Table 9, the phage GJ19P1 has strong lysis capacity on 9 different Xanthomonas pathogenic variant strains of 184 strains, and the lysis rate can reach 91.8%; and the cracking ability of the strain to the single Xanthomonas pathogenic variant is more than 85%. Phage GJ19P1 has a broad cleavage spectrum.
Example 11 lysis test of Xanthomonas phage GJ19P1 on non-pathogenic beneficial bacteria 5 non-pathogenic rhizobia were selected, 5 non-pathogenic bacillus subtilis were inoculated into test tubes containing 3mL TSB liquid medium, and cultured at 37℃for 8 hours at 180rpm to obtain bacterial solutions of each strain. mu.L of the bacterial suspension was mixed with TSB semi-solid agar medium and spread on a common agar plate. mu.L of purified phage GJ19P1 solution (prepared in example 7) was dropped onto a plate, and after natural air-drying, incubated at 37℃for 12 hours, and the results were observed.
TABLE 10 lytic assay of Xanthomonas phage GJ19P1 against non-pathogenic beneficial bacteria
Note that: "+". ++'s completely transparent; "++" medium clear; "+" is slightly clear; non-cleavable is "-".
As shown in Table 10, in this example, none of the phage GJ19P1 identified the 10 non-pathogenic bacteria. The test phage has extremely strong host specificity and no damage to microbial community.
EXAMPLE 12 sterilizing Effect of Xanthomonas phage GJ19P1 in liquid Xanthomonas carpet GJ19 was cultured to logarithmic growth phase, and the culture was dispensed into different test tubes, and the bacterial solution was diluted with an equal volume of TSB liquid medium to a final concentration of Xanthomonas carpet GJ19 of 5X 10 3 cfu/mL. Respectively accessing the final concentration of 2X 10 2 PFU/mL、2×10 3 PFU/mL，2×10 4 PFU/mL、2× 10 5 PFU/mL、2×10 6 PFU/mL Xanthomonas phage GJ19P1 (Xanthomonas phage GJ P1) (prepared in example 7). A control group and a blank group were simultaneously set, and the control group was given a final concentration of 1x10 3 cfu/mL Xanthomonas phage GJ19P1; the blank group was given an equivalent amount of physiological saline. The treatments were cultured at 28℃with shaking at 150rpm, and after 4 hours the residual amount of Xanthomonas carpet was measured. The detection method comprises the following steps: after diluting each treated sample with sterile water, 100. Mu.L of the diluted solution was spread on a TSA solid plate, and the number of colonies on the plate was counted after culturing at 28℃for 24 hours. Xanthomonas carpet = colony number on TSA plate x dilution x 10.
TABLE 11 sterilizing Effect of Xanthomonas phage GJ19P1 at different concentrations in liquid
As can be seen from Table 11, xanthomonas phage GJ19P1 had a final concentration of 1X 10 2 When PFU/mL is carried out, the growth of xanthomonas carpet grass GJ19P1 in a liquid culture medium can be well controlled; when the final concentration of the xanthomonas phage GJ19P1 is more than or equal to 10 3 When PFU/mL, the killing rate of the carpet grass xanthomonas can reach more than 97%.
Example 13 preparation of compositions of Xanthomonas phage GJ19P1 at titers of 2X10, respectively 9 PFU/mL stock solution of Xanthomonas phage GJ19P1 (Xanthomonas phage GJ P1) and Xanthomonas carpet phage YHC5 (Xanthomonas axonopodis phage YHC), 2 strains of phage were mixed 1:1 by volume uniformly in SM solution to prepare a 1:1 composition (composition 1).
Respectively taking the potency as 2x10 9 PFU/mL stock of Xanthomonas phage GJ19P1 (Xanthomonas phage GJ P1) and Pseudomonas syringae kiwi pathogenic variety phage PSA-P1 (Pseudomonas syringae pv. Actinidiae phagePSA-P1) were mixed uniformly in equal volumes to prepare a 1:1 composition (composition 2).
Respectively taking the potency as 2x10 9 PFU/mL, xanthomonas phage GJ19P1 (Xanthomonas phage GJ P1), solanaceae Ralstonia phage GP3 (Ralstonia solanacearum phageGP) stock solution, and mixing 2 strains of phages uniformly at an equal volume of 1:1 to obtain composition (composition 3).
Respectively taking the potency as 2x10 9 PFU/mL stock solutions of Xanthomonas phage GJ19P1 (Xanthomonas phage GJ P1), xanthomonas carpet phage YHC5 (Xanthomonas axonopodis phage YHC) and Pseudomonas syringae kiwi pathogenic variety phage PSA-P1 (Pseudomonas syringae pv. Actinidiae phage PSA-P1), 3 phages were homogeneously mixed in equal volumes 1:1:1 to prepare a composition (composition 4).
Respectively taking the potency as 2x10 9 PFU/mL stock solutions of Xanthomonas phage GJ19P1 (Xanthomonas phage GJ P1), xanthomonas carpet phage YHC5 (Xanthomonas axonopodis phage YHC) and Solanaceae Ralstonia phage GP3 (Ralstonia solanacearum phage GP 3), 3 phages were homogeneously mixed in an equal volume of 1:1:1 to prepare a composition (composition 5).
Respectively taking the potency as 2x10 9 PFU/mL stock solution of Xanthomonas phage GJ19P1 (Xanthomonas phage GJ P1), pseudomonas syringae kiwi pathogenic variety phage PSA-P1 (Pseudomonas syringae pv. Actinidiae phagePSA-P1) and Solanaceae Ralstonia phage GP3 (Ralstonia solanacearum phageGP 3), 3 strains of phage were homogeneously mixed in an equal volume of 1:1:1 to prepare a composition (composition 6).
Respectively taking the potency as 2x10 9 PFU/mL stock solution of Xanthomonas phage GJ19P1 (Xanthomonas phage GJ P1), xanthomonas carpet phage YHC5 (Xanthomonas axonopodis phage YHC), pseudomonas syringae kiwi pathogenic variety phage PSA-P1 (Pseudomonas syringae pv. Actinidiae phage PSA-P1) and Solanaceae Ralstonia phage GP3 (Ralstonia solanacearum phage GP 3), 4 phages were homogeneously mixed in equal volume of 1:1:1:1 to prepare a composition (composition 7).
Respectively taking the potency as 2x10 9 PFU/mL Xanthomonas phage GJ19P1 (Xanthomonas phage GJ P1) and 50% final concentration of ambam 700-fold solution were mixed uniformly in an equal volume of 1:1 to prepare a composition (composition 8).
EXAMPLE 14 sterilizing Effect of Xanthomonas phage GJ19P1 composition in liquid Xanthomonas carpet to logarithmic phase of growth, split charging into different test tubes, diluting the bacterial liquid with equal volume of TSB liquid Medium to a final concentration of Xanthomonas carpet GJ19 of 5×10 3 cfu/mL, to which the compositions of Xanthomonas phage GJ19P1 prepared in example 15 were respectively added, so that the final concentration of each composition was 2X10 3 PFU/mL, control and blank groups were set simultaneously, the control group given a final concentration of 5X 10 3 cfu/mL Xanthomonas carpet GJ19; the blank group was given an equivalent amount of physiological saline. Each treatment was cultured by shaking at 150rpm at 28 ℃,after 4 hours, detecting the residual quantity of xanthomonas carpet grass GJ19, and the detection method comprises the following steps: after diluting each treated sample with sterile water, 100. Mu.L of the diluted solution was spread on a TSA solid plate, and the number of colonies on the plate was counted after culturing at 37℃for 24 hours. Xanthomonas carpet = colony number on TSA plate x dilution x 10.
TABLE 12 Sterilization effects of different concentrations of Xanthomonas carpet phage GJ19P1 compositions in liquids
As can be seen from Table 12, each of the compositions of Xanthomonas phage GJ19P1 had good killing effect on Xanthomonas carpet. The xanthomonas phage GJ19P1 can be combined with other substances to control bacteria, and has no antagonism to other substances.
The combination of the embodiment is not limited to 700 times of the ambam, and can be chemical bactericides such as polyoxin, flumorph, dimethomorph, prochloraz, difenoconazole, flusilazole, myclobutanil, mancozeb, thiophanate-methyl, carbendazim, chlorothalonil, polysaccharide fruit and the like. The embodiment can also be combined with other chemical disinfectants to play a role in preventing and killing.
EXAMPLE 15 control of Xanthomonas phage GJ19P1 and its composition on citrus canker with final concentrations of 2X 10, respectively 4 PFU/mL、2×10 5 PFU/mL、2×10 6 PFU/mL Xanthomonas phage GJ19P1 (Xanthomonas phage GJ19P 1) was homogeneously mixed with equal volumes of 700 times the final concentration of 50% ambam (chemical sterilant) to make 1:1 of compositions 9, 10 and 11.
480 citrus trees were randomly divided into 8 groups (phage 3 group, composition 9-composition 11, control group and blank group) after 1 month of adaptation culture, 60 per group, phage experiments were given 2×10 doses, respectively 4 PFU/mL、2×10 5 PFU/mL、2×10 6 PFU/mL of test phage (prepared in example 7) and 2X 10 5 cfu/mL Xanthomonas carpet GJ19; composition experimental groups each composition was administered 2 x 1 separately0 5 cfu/mL Xanthomonas carpet GJ19; control group was given only 2×10 5 cfu/mL Xanthomonas carpet GJ19; the blank group was given an equivalent amount of physiological saline, 1L of the blank group was inoculated by a stem infusion method, and the incidence of citrus canker was counted for 15d from the inoculation, and the incidence = the number of diseased plants/total number of plants x 100%.
Table 13 influence of Xanthomonas phage GJ19P1 and compositions thereof on citrus canker
As can be seen from Table 13, the citrus trees in the control group had 100% of the disease incidence of citrus canker at 15d inoculation. In each experimental group of phages, the higher the concentration of Xanthomonas phage GJ19P1 is, the lower the incidence of citrus trees is; xanthomonas phage GJ19P1 at a concentration of 10 6 At PFU/mL, the disease incidence of citrus trees is kept within 10% after 15d of virus attack. The Xanthomonas phage GJ19P1 and the composition thereof can be used as a biological bactericide, and can effectively prevent and treat citrus canker.
EXAMPLE 16 preparation of kit for Xanthomonas phage GJ19P1 and its composition and use kit containing 5-10 mL of the kit with 1X 10 titer 7 PFU/mL Xanthomonas phage GJ19P1 liquid or Xanthomonas phage GJ19P1 composition, 1LTSB semi-solid medium, 1L TSA medium.
The using method of the kit comprises the following steps: taking the potency to be 1 multiplied by 10 7 PFU/mL Xanthomonas phage GJ19P1 liquid or Xanthomonas phage GJ19P1 composition, the split spectrum of the test phage was determined by double-layer plate drop method. And selecting single bacterial colony to be detected, inoculating the single bacterial colony to be detected into a target liquid culture medium, and carrying out shake culture under the condition of combining the growth characteristics of the strain to be detected at the target temperature to prepare bacterial liquid of the strain to be detected. Mixing 300 μl of strain suspension to be detected with 5mL of TSB semi-solid medium, spreading on TSA plate, and collecting 10 μl of Xanthomonas phagemidThe body GJ19P1 liquid or the composition of Xanthomonas phage GJ19P1 was spotted on a plate. And (3) culturing at a target temperature according to the growth characteristics of the strain to be detected after natural air drying, and observing the result.
Example 17 application of kit of Xanthomonas phage GJ19P1 and composition thereof to citrus canker
The main component of the kit 1 is 5-10 mL with the titer of 3X 10 8 PFU/mL Xanthomonas phage GJ19P1 liquid.
The main component of the kit 2 is 5-10 mL with the titer of 3 multiplied by 10 8 PFU/mL Xanthomonas carpet phage YHC5 liquid.
The main component of the kit 3 is 5-10 mL with the titer of 3 multiplied by 10 8 PFU/mL liquid of P.syringae kiwi pathogenic variant phage PSA-P1.
The main component of the kit 4 is 5-10 mL and the titer is 3 multiplied by 10 8 PFU/mL liquid of the Solanaceae Ralstonia phage GP3 (Ralstonia solanacearum phage GP 3).
The main component of the kit 5 is a 700 times solution of the ambam with the titer of 5-10 mL and the final concentration of 50%.
The main component of the kit 6 is 5-10 mL with the titer of 3X 10 8 PFU/mL Xanthomonas carpet phage GJ19P1 liquid, 5-10 mL titer of 3X 10 8 PFU/mL carpet grass xanthomonas phage YHC5 liquid, 5-10 mL titer is 3X 10 8 PFU/mL liquid of pathogenic bacterial strain phage PSA-P1 of Pseudomonas syringae and kiwi fruit, 5-10 mL of liquid with titer of 3X 10 8 PFU/mL of Solanaceae Ralstonia phage GP3 liquid, 5-10 mL of ambam 700 times liquid with final concentration of 50%.
The test process comprises the following steps: the citrus trees are randomly divided into 8 groups (a kit 1 group to a kit 6 group, a positive control group and a negative control group) after being adaptively cultured for 1 month, and 20 groups of the citrus trees are selected from each group. 1L of the final concentration is 1x10 respectively by adopting a stem transfusion method 3 cfu/mL of the pathogenic variety ACCC 03526 (Xanthomonas axonopodis pv. Citri) of xanthomonas carpet citrus is used for carrying out toxicity attack treatment on 140 citrus trees in the kit 1 group to the kit 6 group and the positive control group, and the negative control group is used for carrying out stem transfusion on 20 citrus trees 1L of physiological saline was respectively introduced. Three days after the virus attack treatment, the stem transfusion method is adopted to inoculate 1L of thousand-fold dilution liquid of 1 group of the kit to 6 groups of the kit to 5 groups of citrus trees, and the positive control group and the negative control group are respectively given with the same amount of physiological saline. The incidence of citrus canker was counted 15d from inoculation, incidence = number of diseased plants/total number of plants x 100%.
Table 14 Effect of kit of Xanthomonas phage GJ19P1 and its composition on citrus canker
As shown in table 14, the lower citrus incidence of the kit 6 group than the kit 1 group, indicated that the kit 6 containing the composition was significantly more effective in controlling citrus canker, and the kit 6 containing the composition was better in controlling citrus incidence than the kit 1 containing only xanthomonas phage GJ19P 1.
Example 18 application of kit of Xanthomonas phage GJ19P1 and composition thereof to bacterial leaf streak of rice
The kit of example 17 was used for each of the kit 1 to the kit 6.
The test process comprises the following steps: 160 rice plants were randomly divided into 8 groups (kit 1 to kit 6, positive control group and negative control group) 20 plants each after 1 month of adaptation culture. 1L of the final concentration is 1x10 respectively by root irrigation 3 cfu/mL of yellow monad rice fine streak pathogenicity ACCC03523 (Xanthomonas oryzaes pv. Oryzicola) is used for carrying out virus attack treatment on 140 rice plants in the kit 1-6 and positive control group, and 1L of physiological saline is respectively applied to 20 rice plants in the negative control group by a root irrigation method. Three days after the detoxification, 1L of each of the thousand-fold dilution liquid of the kit 1 to the kit 6 is inoculated by a root irrigation method, and the positive control group and the negative control group are both given with the same amount of physiological saline. Counting the bacterial nature of rice within 15d from inoculationIncidence of leaf streak disease = number of diseased plants/total number of plants x 100%.
Table 15 Effect of kit of Xanthomonas phage GJ19P1 and its composition on bacterial leaf streak of Rice
As shown in Table 15, the incidence of rice plants in the kit 6 group is lower than that in the kit 1 group, which indicates that the kit 6 containing the composition has remarkable control on rice bacterial leaf streak, and the kit 6 containing the composition has better effect on controlling the incidence of rice plants than the kit 1 containing only Xanthomonas phage GJ19P 1.
Example 19 application of kit of Xanthomonas phage GJ19P1 and composition thereof to bacterial wilting disease of cassava
The kit of example 17 was used for each of the kit 1 to the kit 6.
The test process comprises the following steps: the cassava strains 160 were randomly divided into 8 groups (kit 1 to kit 6, positive control group and negative control group) 20 strains each after 1 month of adaptive culture. 1L of the final concentration is 1x10 respectively by root irrigation 3 cfu/mL of Xanthomonas carpet cassava wilt disease-causing variety ACCC 03517 (Xanthomonas axonopodis pv. Manihotis) is subjected to virus attack treatment on 140 strains of cassava in the kit 1-6 and positive control group, and 1L of physiological saline is applied to 20 strains of cassava in the negative control group by root irrigation. Three days after the detoxification, 1L of each of the thousand-fold dilution liquid of the kit 1 to the kit 6 is inoculated by a root irrigation method, and the positive control group and the negative control group are both given with the same amount of physiological saline. The incidence of bacterial wilting of cassava within 15d from inoculation was counted, incidence = number of disease plants/total number of plants x 100%.
Table 16 influence of kit of Xanthomonas phage GJ19P1 and its composition on bacterial wilt of cassava
As shown in table 16, the lower cassava morbidity of the kit 6 group compared to the kit 1 group, indicates that the kit 6 containing the composition was significant in controlling bacterial wilting of cassava, and the kit 6 containing the composition was better in controlling cassava morbidity than the kit 1 containing only xanthomonas phage GJ19P 1.
Example 20 application of kit of Xanthomonas phage GJ19P1 and composition thereof to mango bacterial black spot
The kit of example 17 was used for each of the kit 1 to the kit 6.
The test process comprises the following steps: after 160 mango trees are adaptively cultured for 1 month, the mango trees are randomly divided into 8 groups (a kit 1 to a kit 6, a positive control group and a negative control group), and 20 strains are selected from each group. 1L of the final concentration is 1x10 respectively by adopting a stem transfusion method 3 cfu/mL of Xanthomonas campestris strain ATCC 11637 (Xanthomonas campestris pv. Mangifetaeindiae) is used for carrying out virus attack treatment on 140 mango trees in the kit 1-6 groups and the positive control group, and 1L of physiological saline is respectively applied to 20 mango trees in the negative control group by a stem transfusion method. Three days after the virus attack, 1L of thousand-fold dilution liquid of the kit 1 to 6 is respectively inoculated by adopting a stem transfusion method, and the positive control group and the negative control group are both given with the same amount of physiological saline. The incidence of mango bacterial black spot within 15d from inoculation was counted, incidence = number of lesions/total number of lesions x 100%.
Table 17 influence of kit of Xanthomonas phage GJ19P1 and its composition on mango bacterial black spot
As shown in table 17, the lower incidence of mango in the kit 6 group than in the kit 1 group, indicated that the kit 6 containing the composition was significantly more effective in controlling the incidence of mango bacterial black spot than the kit 1 containing only xanthomonas phage GJ19P 1.
Example 21 application of kit of Xanthomonas phage GJ19P1 and composition thereof to kiwi canker
The kit of example 17 was used for each of the kit 1 to the kit 6.
The test process comprises the following steps: 160 kiwi trees are randomly divided into 8 groups (a kit 1 to a kit 6, a positive control group and a negative control group) after being subjected to adaptive cultivation for 1 month, and 20 strains are selected from each group. 1L of the final concentration is 1x10 respectively by adopting a stem transfusion method 3 cfu/mL of Pseudomonas syringae actinidia pathogenic variety ATCC BAA-2502 (pseudomonadsysingae pv. Actinidiae) is used for carrying out toxicity counteracting treatment on 140 kiwi fruit trees from the kit 1 to the kit 6 and the positive control group, and 1L of physiological saline is respectively applied to 20 kiwi fruit trees from the negative control group by a stem infusion method. Three days after the challenge, 1L of thousand-fold dilution liquid of the kit 1 to 6 groups of the kit are respectively inoculated by an injection method, and the positive control group and the negative control group are both given with the same amount of physiological saline. The incidence of kiwi fruit canker was counted within 15d from inoculation, incidence = number of diseased plants/total number of plants x 100%.
Table 18 influence of kit of Xanthomonas phage GJ19P1 and its composition on kiwi fruit canker
As shown in table 18, the incidence of kiwi fruit in the kit 6 group was lower than that in the kit 1 group, indicating that the kit 6 containing the composition was remarkable in controlling kiwi fruit canker, and the kit 6 containing the composition was better in controlling incidence than the kit 1 containing only xanthomonas phage GJ19P 1.
Example 22 application of kit of Xanthomonas phage GJ19P1 and composition thereof to ginger bacterial wilt
The kit of example 17 was used for each of the kit 1 to the kit 6.
The test process comprises the following steps: ginger 160 strains were randomly divided into 8 groups (kit 1 to kit 6, positive control group and negative control group) 20 strains each after 1 month of adaptation culture. 1L of the final concentration is 1x10 respectively by root irrigation 3 cfu/mL of Solanaceae Ralstonia ATCC11696D-5 (Ralstonia solanacearum) is used for carrying out toxicity attack treatment on 140 ginger strains in the kit 1-6 groups and the positive control group, and 1L of physiological saline is respectively applied to 20 ginger strains in the negative control group by an injection method. Three days after the challenge, 1L of thousand-fold dilution liquid of the kit 1 to 6 groups of the kit are respectively inoculated by an injection method, and the positive control group and the negative control group are both given with the same amount of physiological saline. The incidence of ginger bacterial wilt in ginger for 15d was counted from inoculation, incidence = number of diseased plants/total number of plants x 100%.
Table 19 influence of kit of Xanthomonas phage GJ19P1 and its composition on ginger bacterial wilt
As shown in table 19, the lower ginger incidence of the kit 6 group than the kit 1 group, indicates that the kit 6 containing the composition was remarkable in controlling ginger bacterial wilt, and the kit 6 containing the composition was better in controlling the incidence than the kit 1 containing only xanthomonas phage GJ19P 1.
In conclusion, the Xanthomonas phage GJ19P1 (Xanthomonas phage GJ19P 1) and the composition thereof have high safety, can be used as an active ingredient of a preparation kit and a biological disinfectant or biological pesticide, and can be used for preventing and treating but not limited to various bacterial diseases caused by Xanthomonas.
According to the embodiments 17-22, it can be seen that the single use of the Xanthomonas phage GJ19P1 has a killing effect on bacteria used in the attack experiment, and can play a remarkable role in preventing and controlling diseases of attack plants, but the composition containing the Xanthomonas phage GJ19P1 has a better effect in preventing and controlling diseases of attack plants.
The present embodiment is only for explanation of the present application and is not to be construed as limiting the present application, and modifications to the present embodiment, which may not creatively contribute to the present application as required by those skilled in the art after reading the present specification, are all protected by patent laws within the scope of claims of the present application.
Sequence listing
<110> Fei Ji Leke (Nanjing) Biotech Co., ltd
<120> a cross-species cleavable Xanthomonas phage and compositions, kits and uses thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 34696
<212> DNA
<213> Artificial sequence (Artificial Sequence)
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gccaacgttg cgcggcaggt agatgttcgg ttcggtactc acggcgtgtt acctccgggc 120
ttggtgatcc acagcgtgaa gcatgggatc aggttgatgc accagcgctt gttgtgcggc 180
gagtagtgca cgccgatcca agtgctgtgc ttgttccgca gccatcccca cttccactgc 240
ggcgcgtcgt tgcggccttc gatgtacgca gcgttgaccg catcccgcac aaccgatgcg 300
aggtagttgg cctgactact ggacaccgtg tcgaatcctt tctggatgcc gggcaggaag 360
actcgatgcc cgtgtacctc tactttcgtg ctgctcatgc tgcctcctga tagaagcggg 420
cacgctctgc atccatcatc acttcttccc gaacgtcacg cttaccgcct gcgcggtgca 480
gcttgttctt cgaggtgccg atgtagcgga agcgttccat cgccgggtcg ttgagcttgc 540
cgcccatgat gatgaagtcg catgcacctt gcttgcccgt cttgctgtcc ttcagtgcgg 600
actgcggcgg gtactgcaca ccctcaccct cggcgctgat ctgcgaagtc gccacgccaa 660
gcagagcgcg caggttcttc gccaacgtgg gcagtcggaa gtggatgccg atgtcgtgct 720
cgtccatctg catcagttct tcgggcgtct cctgcaccag aggcaggcga gccttgcgat 780
ccatgcggtc ggtgatcgtc tcggccaacc cggcaagctc gtcgcgcagg ctgaactcag 840
cgccaccatt ccactgcacg atcttgctga ccgcagtggt ggcaagctcc aactccaaca 900
gcttctcgat catgccctgc tctgcctcgg gcggaacagg atcgcgcatc tgcttcagca 960
gaactttcag cagggacttc ttgtcgtcgg gctgcgtctt ccacttgaac tccatggccc 1020
agccatcgaa cacatcgaac ggcaagtcct tcgtgtccgg gaactccttg tagtacgcac 1080
gcaacgcatc gagcatcagc acggtcagct cgtccagccc atcacgtggt gcactgggca 1140
cgagccggtc gaacttgcca cggttggtgc acaaccgcag cagcgtggtg tcaagactca 1200
tcaggttcct cctcgtgttc gtacttcgtt ggtgcgagct gctggtagat cgagttcttc 1260
gtctcgaacc tgcccggctc tttgacgaag accaccactg acgtgatgca gtcctcgccg 1320
ttcagctctt gtcggtaatg cccggtaact ccggacacca tcgcgcggaa gcctacggtt 1380
ggcgggcgtc cgatgtgatc cttgtagtac acaactttct tctcagccat gaagcttctc 1440
ctctatgtaa tcggtcgggt aatacttcgg gtcacgtggt gacgtgatgt tcctgtagtc 1500
cagaccaaac gcacgtagac gcgcagcgat tactcgcgcg ctgtcttggc cgggattgct 1560
accgttgctt ctacctctgt cgtcatcgag ccacgtcgct acacgttggc ctcgatcagt 1620
cagctgcttc aacaccttgt tgtgcagctt ggtgccgagc aacgaccacg cttctgtgca 1680
ctggcccacc ttgaaggcgg atagcgggtc ttcgcatagc acgatggtgt cccccttgcc 1740
cacgccgtag cgtgcgacaa gcccatcctt cggcacatca ggagtcaacc atttgggttg 1800
acggcttgtg cttcgtgctg tccagtacac aacgtcgtcg ccttcagtga tcggtagtac 1860
cacccgcccg atgctcgggc accagtacag ccgcagcttg tcgatcatct gaagcgagag 1920
tcccatcttg aagaaccaca ccttggcgtc gagcggccac tcgcgggtgt cgtatacccg 1980
tggctctggc aactcaacag ttgcgcgcac tctgcgctcg ctcgtttgct cggcgtggat 2040
tcgtgcaagc ttggcctcga tactctcgtg ctcacgcttg aatcctgtgc caccgcacct 2100
gaaacagaat gcggtggact tgtcatgctc acgagtaatc agcaagggcg taccctcccc 2160
gcactcatgg gccactcgtg tgcggccacc aacaggcagg cgctcggcgt gtggtagcca 2220
gtcgttgtcg ggaagggtat cgctcatgct atcctcgtcg gatcagtagc tccagatgga 2280
gccgggcgtg gcgaaggaca cctcgccggt gctggtgttg accagctgac gattgccctt 2340
cttgtcggtc acgttggtga ccagcaggta ggtgtcgccg tcgatgactt cgcctgcgcc 2400
ggagctgatc gcccggtagt tgaagccgac cttcaggtca cggcctgcgg tgaactggct 2460
gcggctcggc gcgctggtcg gcggcaccgc cgggctggtg ctgccccggc tcacgactga 2520
ccgccttcac cgcccgcagc cttggcggca gcggcttcgg cggcgaggcg ctgggcctcg 2580
gcaacggcgg cgtcgatttc ttcctgcgcc ttcagcactt cctcggccga catcagcagg 2640
gcgctgctgt cgatcaggtt cagctcggca tccagacctt cgccggtctg caccttcagt 2700
tgcagcacgc ccttgtcgtt cttgccggtg gcgacgatca cgccggacag gacacgcttg 2760
ttcagggcac gaccgaacag gtaggacacg gcgtcgccgg acttcagcgc ttcgatggcg 2820
gcgctgttgg cctcgacggc atccagcttg gcgagcttct cgttcagcgc atcgaccttg 2880
gcctgcgcct tggcgatgtc ttccaccagc tcggcgcgct tgacttcgta gtggctgagg 2940
gtggtctgct cggtggtgtt ttcgttcgac atgcggtgta tctcctttca ttgacgggtt 3000
gatgggtggt ccgtaacgtg gtcccttccg gcacgcaatc agtgcgagat ttactggaag 3060
atcagctcgc tgttgtaccc gccgccgcca ttgtagaagc gacggcgcag cacgctctcc 3120
accacttgcc gcctatcata gctcactgct agcttagtgt caagcgtggt tcggtagctc 3180
ctcgcatccc ttaccagtga ctcccatgta cccttagccc acttgttggc ggcgccacta 3240
cggatcacgt cacttgactt gcgttgcagc ttactaccat tgcggccgtg tgtcaaccat 3300
tggcctagcg ggtagctaac ttatcagctc gacagagtac cgatagaccg gtgttcaggt 3360
tgagccgact gtgtgtgcta taggtgtacc aaaaggccga aacgatacag atggccctca 3420
cacgcctaca gctgcggttc gcatggtagc aggtagggta gcccctgtca agccccttgc 3480
ggccctcctg aagccctgtc gctgccccag tgacgccatt ggatcacacc acgacggcca 3540
cgtcaacacc tttcggcgag agatttatta accattcagc caaggcagca cggtccagcc 3600
atacaggcag caggcgcacg cccacccgca ggcacccacg cacggccgca caggcgcacg 3660
cacacgccac gcgcaggcag gcacaggcac acacccacgt gcattgactc gcgcccgcag 3720
gtgaggcgca caggcgcgca cacgcaggca cacccgccca cgcacccacg cgcgtcaggc 3780
gcaggggcgt atgggggcac gcgcgggagc ttctattgga gtgaccctcg cgtaattgtg 3840
aagcaaattt tggtccggct acccacgagg ccctatgact aagccttgcg aactggtgcc 3900
tttagcttta cgataggccc ggagctgtac acgtcgtgag ctatcgcagc agccatggcc 3960
ttagccacag acttgctcgc atgataggcc cctagagcta gcgccttgcc actgccatcg 4020
aagtacggag cctcctcgac ccactgctgc ttaccagtag tgaccgtgag tgcgccgtcc 4080
ttaaccacaa gcaccgttgc ggacttgcct acgtgctcca tgtagtcttc gcccgcttta 4140
agcgcagcga gaaccgggcg aagcactgtg aaatccccgg tgacacccag cagcactggg 4200
ccggtgggtg tcgagacagg gaggattttg cggacggtgt gcttgtggtc gccttgtgtg 4260
gcctgactat ccgcccacag gtgcttgccg tcccacgcta tcgtggtcat cggtcacccg 4320
tacaccggcg cttgccggta ccgcacacct cttgcttcca cgcacggaga gcggccttgt 4380
ccttgttgca cagaacggtg tccatgcggc ggtcttgcgc gagcttgacc accttgtcct 4440
gactggaagc ggagccgcgc tcaccaccaa ggtaggtgat gtggcagtcc ttcagcagat 4500
catcactggg aaccggcagg ttcgcgcagg aggtcagcga catcagcagg aacagtgccg 4560
tcacgatact cgggaacggc gtcgagagct tcttcagtgc gaacacgcac gtcctccttc 4620
tctttgatat aggtcgcgta cccggtggcc tgcgtctgcg cagccttggc ggtagcggtc 4680
gtgccgactt gggcagtttc cagcttctcc accttgcgtt gcaggccgta gacgtacagc 4740
ccgaagcaca cgaacatggt cacgaccagt agcaggccga tagccttgcc gctgagacgc 4800
gcgatcactt cacttggggt ttccacggtt ggatcacctc gttgttgggg cgcaggcaga 4860
ggtcaaactc tgcgtcgcgt cgcttgggaa tgccagagca ccggctactg gacagtcggc 4920
agtcgagctt accgacgtag acccagcgca ggaactcctt gcagctacct tcacgatccc 4980
ctgcgttcgc cttgcgcagc atggtgctgt ctgcgaacgc cttctcgccc acgttgaaca 5040
cgaagtccgt gtaggcgtcg atctggttgc ggttaacggg aaccttcagg tgcttgcgca 5100
cgccctgctg ggcgatctgt aggtcttcga tcagccactt gtcgcactga gccttcgttg 5160
ccgtcatgcc catcgctacg cccttggtgt ggccgtagca gatggtcggc acccggccgg 5220
tgtcgaggta cgcagcgtag cgtacccctt cgtggccttg ggtgaatgac agcagcgttg 5280
ccgaaatgcc gatgaagatg ccgccgacga ggccgaacag ccctttgttg ctggtatctt 5340
ggttctgctg ctggttcatg gttgctcctt acggggtcag cgggtcatcg tccgggtcca 5400
gatcgaccgg agtgtgcggg ccggagttcg gacccgagga ctgcgtgcgc gcaccagcag 5460
cgatcagggc gtcgagcacg agggtcgcgt ccttctggaa ctgggcgagc agctgcgctg 5520
cggttgcggg atgctgctcc gcgtgctcca cgagggctag cagttcacgg tgcaggtagt 5580
tgccacggcc gaacaggccc gtgtagttgc tggtgaagtc ggacttcaac attggttacc 5640
tccggtaacg gtcaagtagg gatggacggc cacgttgcag tggactcttg cgggtcgctg 5700
ccttcatgcc ggtaggatcg ttgatccagt cttggaactc tcgttgctgc tgcttggcga 5760
tggccttgtt ctggtcgagc gcgagctgcg caacccagtg acgcacgcta ccttccagtg 5820
catcgagccg gtcatcgtgt gcgatgctgc ccttctgctg cgtgatcttc gccatctggt 5880
ggaacaggct gtagacctga cgcttgccgc tggatgcgta gcgctgggtg tactcgtgat 5940
ccatttcaac cacggattcc agcatgacca gcgagccacg agccatgaca ggttcgagcg 6000
tgccgatgat gcgggcttcc ttgttgccgg tcacgaactc ctcgcggatg tcgcccttgt 6060
atcccttctc ggtaccgcga tcttctcgca gcacgggcag gaacaccttg gcgaacgcgc 6120
cgtagcccat gttcttttcg atgctgatga cgttcggctt gtacttcacc gcaatgcggc 6180
gtagttcgag cagcccgtcg ccatcgtagc caccgggcac agcacccacg gccaacacgt 6240
acacggtgga gttcaggaag gcggtcacgg cgaacgccgt ttcgtctccg ttggcaccac 6300
cacctgccgg gtcgatctgc atgtgcacgc cctgcacggg tgcagtctcg gtgctcatgt 6360
cgtgcggcat catcatgacg aagccgtagc cgctggactg gaacgcgcgc tggtgagcgt 6420
tgctcaggcc gcgcgtgatc gtcaggggga agcggtcgcc gccacccgga atgaccacga 6480
ggttctcggt cttcagcggg aaacgcagcg cgtccatcag ccgcgtgttg agcatgtagt 6540
tcagctggta gctggacggt ccacgctgac gcagttcgga ctggtgcttc tgctcaccgg 6600
ccaacagctc gtcggtgcac tgcccctcgt cacccatcgg gccaccaccg aacgccaaca 6660
tcggatcggc ttcgagccgc tgtacgatag acggtgccag atgcggaccg tagtgctcgc 6720
gctgtgccgg ggtcgggtag cgaccgggcc agatgcgaac gtcgtagcca cgtccgggca 6780
gggtgttgta gatggagtcg ccggtctgcg gcgtgccgag gtacaggata cgccccggag 6840
taccgggctt gccggtgcag atagcggaga actccaacgt gatggtcagc agcagctcgc 6900
ggttcgctgc ggtgcggctg ttcttgttgg actccacgtc atcggcgatc agcaggtctg 6960
gatcttgcgg tgctcgcgcg cgcgccagta gaactgggcg acggcataca gaattgcgag 7020
cgtagttacg atggcagcac catacgtgct gatgaagttg aggtagaccg cgccgagcgt 7080
aagcggcgag gttcggacga cctcattgac cacactcggc ggggtcatgc tgttctcctg 7140
taggttaagg ccagtcgtcg ggattgccga cgagcttcca ttgcaggcgg ttgacgttgt 7200
tcatgccaac cgcgatcagg tcaccgggga ttccggtggc gatcacgtgt tcggttgcga 7260
agcgctcgcg ctgtgcacgc acgcacaagc tgctcccgtt cttataggaa agcagtacgt 7320
cgctctgcgt ggcgaatagc gctcgcttct cgtccatgca cagccggggc gtggttgcac 7380
cggggatcac catggtctgc atcgagctag ccaccgtgtc gtaccagtag aacttgcaca 7440
cacctcccgc catgtaggca atgtgcgggc gcatggtctg gtcgaatgcc agtgcgatct 7500
gcgtgatgcc cgagtccgtc agcacggtga caggggcaac gccgtcgcgg taaacgacga 7560
cgctgctgcc gtcactctcg gcggtccaca gcgcagcttg gaggtcatcc actgctgcgt 7620
tgagggatag cccgccctgc tcgaagtcaa tgagcgggcg ggtgcgccgg ttctgctgct 7680
ctacccagtc accacgttgg atgctttggg agacagtagg gatcatggcg tgtaccggct 7740
ccacgaaacg cgcatgcgca ggttcgccgt ctccgatccg gtcttggtga cacccggatt 7800
gaaaccaact tggaacggca ccgcagagtt cagtccctgc gcggcagtga tcggacccgg 7860
aggaacgttg ttgatcgaca tacggcagac cacatcgcgg tagtaggtac cgggaacgta 7920
gggcacgtac tgaatctgca agtccgagaa gctggtcaga gtgcccggcg cgtactggtc 7980
gaaggaatgg aaggagccag aaatacgagc cgtctccggg tccggtgtgt agtcctttcg 8040
tatagagcgg atgtcgaacg aaacgaaaga gcttccgtac atcgtggact cgggatcagc 8100
cgcgtagtct tgccgagagc tgcggagttc ccagcccacg aacgaggaac caacactggc 8160
ggagtccatg ggcgggagca tgtcgatgtt gctgtaccca gcgaagctgc tggtaagcac 8220
cgtagcatcg aactggtaga agctgtagag cttcgacgta acgtagatgc cctgctcctc 8280
gatacgcccg gaccagtagg ccacacggtt caacccgtcc gtgaccttga tgctcaagaa 8340
gtaggagctg tcgtcattct gacccagcac gtacttgtgg ttccacttaa cggtgtccac 8400
ccacagaggc ttgttctgct tgctgcggaa tgcggacagt accacctgca cgtacttcgt 8460
gtccttgggt gctgcacctt cggcgaccga ctggtgccat gcgccgttgc tgcccgagga 8520
aaccatgttg ccccagttgc gttgcagctc ggtcccatcc tccttgcggt agatgatgga 8580
cacgcgcgca ccggccttgc ccttggagct tgcgccctgc tggacttcgg cggtgcaccg 8640
gatgtagtcg ttgaccttgg cgggaatcag cgggttgagt agatcgctgc cgcccgtctt 8700
gatgttggcg aagcgcgcgc tgtatacgcc gctgtcggtg tcgtagtccg agccggtgcc 8760
aatcgaccag ccttcgccgt tcgatccacc gcccggcaac cacgtgccgt cgtcgcctgc 8820
ctcgaagtcg ccgttaggca cgagggtttc ctcgtccacg acttcagtga aagccgccca 8880
cttgacgacg acttccttgg tgatgttgtc cacccgtacc gcagcgcccg aagggagcgt 8940
cgattcgatg acctccacgg tgcagcggcc gactgggttg ttgatggaca ggcgagactc 9000
gtaagcctcg cccgccgtgt acccttccag cacgcccgtc accgacaggc catctgcaac 9060
ctcgtccgcc atcagcggtc ggccgtacag cctgaagtga ttagccatgc ttactcctta 9120
gccaagtcgc ccgagagcaa ccacgtgttg gtgtcgccgt cgatgcacgt tgcagtgacg 9180
acggagttgg tgccgcgagt tgcggcggtg cagccgaccg ggacttcgag ggtcacgccc 9240
acgtcgccag tgatgaccac cgggcctgcc ttctgctgca cgctgaagta gctgcccgag 9300
gcgagcttcg tctcaccgtc acccacttcg gggatcaggt tgatgttgac ggcgcagcct 9360
ccggcatacg tgcagcgcac gaggccgtat tcggtagcct tcgcggcgta gtaggtcggg 9420
ccttctgccg ggatgatcgg cacgcgcagg ctcgcagcag tgattggcgc aggcccggtg 9480
tagggcttgg cgtaaccacg cagcacggcg aaccagatgc tgccctcggc cggaaccgtg 9540
gcgaagcgga tgaagctggg atcggtgccg tcggccagcg tgacctgata gtccacgtcc 9600
ggctgcatgc cgataccgtt gacgtacacg tcgtagaagc cagcgccgga cacgtccgcg 9660
cccaccatat cgaagtcggt gtcgtccact tgatcgccgg tccatgcacc ggaccagacg 9720
cgcggggcga cggacacgat accaccagcg gccagcaact cggtgacctg cttgtagatg 9780
ttctggataa tctcgttcag gtccacgatc acgccgacct gctggtcgat gctaagggtg 9840
gcgatgttga gcgagtcgaa catttcctga atgatcttca ggagctggtc gttgctgagg 9900
ttaaggttgg tcttgctgat gatgctaccg ttaacgtagt caacgagcat ctgctgcacc 9960
ggagtgctgc ggtagatgta gagcttgcgc ccagcggcga cggcaggcga gacacggacg 10020
gtactagcgt catcgtcttc gctcaggact tccacggtgt gtggggtccg gtcggtcagc 10080
agctgagtct cctcgtccag cagtccgctc atagcgtaca cgtgggacgg gttgatgtag 10140
ccaccggtaa agctgatctg ccagtcagtg gtcacgccat cggcgtcgaa cacgtctgtc 10200
gcgtagcgca gctgcgggtc tggaactgcc atgctgcctc ctgtttgcta taggtgtacc 10260
aaaagggtgg gccatccttg gccctgattc tcactcctcc ttccactggc tggccgcgcc 10320
cttcagcagc gggatcacca tgggcagtcg cccgcccgga atcacgtcgg ccagattctc 10380
cggcttgcca gcgatgccct gcgcgaagtc gttgacgacg cccagcgacg gcgcgaactg 10440
cccgccgatc acctccttgc ggttgccgaa ctgcccaccg gccagctgct tcagccacgt 10500
cggaccctgc gtgtcggtgg cgtactcgta ccagccacca gccacaccgg tgcccacgtc 10560
cagcacgtcg ttgagcatgc ccgtcgccga cacgtagttc agcgtggcct tgcccagcgc 10620
cagaggcgac aggatgttct ggtcgatggc ctcctcgcgc tcctgatcgt tcatcagggc 10680
cacgcgggac agcacgcgca gggcttgcag cggcgcagcc acggccatcg ccgcagcgat 10740
caggcccgca gtctgccacg caccgtagtt acccacgtag cggcccagct gcttctgctg 10800
tgcgaccagc gagaacgtgc ggtgctggaa cagcagcttc agccaaccgt tgtgcgccca 10860
cttgcccgtc tcgccgatga aggtgtcctg aagaatctgc gaggtgccac ggttgatcgc 10920
gttgtagaac gccaaccatg cctgccgatc cgctagtcca tcgacgttgc gcgggtccac 10980
tgcttccagc ttaccgtcag cgccccagcg cacgacctta tcctgcgtcg cccgcaggcg 11040
gttcagcagg ttgtcgtcga tgcccatgtc cttcagggcg gtgtcagcct cggcaccgtc 11100
gcgcaggaac ttcacgccct tggcgatcag cacttccgcg aagccccgct gctgcacggc 11160
gagcacggcc cgctgccccg agaggatgcg gttcgtgtgc gcggccttct gcaccagcga 11220
ggtcaggaag cctgcgcgct cgctgccata catcgacgcc tgcgtgtcca acgagttgaa 11280
catgtgcagg tcgtaaccgg cgaggccgaa gccgcgaccg ctgatgaact cgaagccgtc 11340
gaggatttgg ttgccgctct cctcgccctt caggatgcgc atgatttcgc cacgcatgcg 11400
gggagcgatc cctcggcgtc ttcgtactgc ttggtcacgt cgaggtcgat acgcgccttg 11460
gtgtgcttcg caccgccacg gctgtacttg cccatcacct tctgaatctc ctcctcgttg 11520
agcgacatcg cccgcagagt gtcgcgcagc gtgtccgcct gcgactccga gaagacctga 11580
cccggcgctt cgatgatacc cgcagccttg cgctcgatgc gttccagata cttggtagcg 11640
aacttgtcga ggaagtcgtc cccgtacagc tcgcccatca cctcgaactg atcgcgcatc 11700
gcctgacgga taccctcacg acgaaccgga ccagcagcgg cgatactgcc gcccagccac 11760
gtgcgggact cgtagccctc cacgtcttct tccggcagac gctgtgcgcc caccgtaccc 11820
gccgcgcgct gctctgctgc catgcggttg tagccacggt ccagctcacg ggccattgcc 11880
ttcactgcct cgtgtgcgtc cgggatgtca ccaccgttga agcgcgtctc gcggtacagc 11940
ttcactgcgt actcgaagtc cgcacgtgcc ttccagcccg tggcgaagtc cttgatcgcg 12000
ccgataccgc gcacgtcagc acgccacagc cggtaggccg cttcccactg acgcgggcgc 12060
gtacccatga acgactcgaa ccggccgtgc cgatccatcg ccgcagtgct gtgtcggcct 12120
gctgcaccct cggggttctc caacagggtg accgcaagca tgcgcgcgac cggagacttg 12180
gacgacataa gggtcgtgct cgtagcttca agcccgaagc gctgcatgat ggtggacagc 12240
ttggccgcgt cgatgccgta cttcgcactg aagcgctcgg agcgggccat cacctcagcg 12300
acctgcacgc gcattgcgtc gtcgctaacg gacttgtcca gacccgtgcg ctggtagaac 12360
gccttgcgct cgcggttcga cttgaagatc gagttggcga gcatgccgcg agtctgacgg 12420
gacgacgcct gcgtgtcacg cacttcggca ccgaggttgt caccgaccac ggacatcaga 12480
cgttcgagcg cggactcctg attcttcagt cccatcactc gtgcgatctt gtccttcagc 12540
cattcccacg cattcgggcg ggtgttgccc ggcgcaggca tctgacgcag caccgcctgc 12600
acagcgcggt tggtcaggcc gtaggaaatc agctcgtcgg tgttagcgag gaaccctgcc 12660
tgcggctggt tgatgcggct ctccatggca gcagtggtca gcgtggagtc cacactgccg 12720
atgctgttgt tgatgtgcgt caggaggtct tccatttcct gaccgaacac ggtgttgctg 12780
cgaagctcac gcaaggtagc agcgtgcagc atttcgtgca ccacaacctc gggcgtgtag 12840
ttgcgcattg ccatcgcgtc gaggccgggg ttgtacccgc ctgccacacg gaagccgccg 12900
cccatgctgg aaccaccgaa ccacttgtcg agttcggact ccggcacgat cttcaccgta 12960
tccagtcgcc cgccgcgctt cagcgcattg gcgatggcac gcacgtcctc ggggatcgag 13020
gcgttggatt ccagatactt cagaacgccc tcgacgttgc cgcccttgat gcccgcagtc 13080
ttccacgcac gttccagcag cgggtcatgc tgatccagca cgcctcccac ggtgtcgttg 13140
taccgaccgg tcgaggcgtc cacgttgtcc acgtcacgag cgagtgcgcc agtggtgcct 13200
gcggtggtgg tcgtctcctg cacgtcaggg cgcgcgaaga tgcggaagtc gtccggcacg 13260
tcagccatac cggcgcgcag ccaatccacg gcctcctgct tcgcaagctg gtggactcgg 13320
ttgttgacca gcaccgggtt ggcgttgtcg cccagctcgg ccacagcgcg cgccttcagg 13380
tcagccgacc acgcttcgcc gtaagcggca gcgttgcctg cggacaggtc gatggtggcc 13440
ttgtcccgaa tctgcgcagc cgagcgcacg cccggaatgt gcatcgcggt accgatggtg 13500
gtggcgaacg cggcgtcggt catgaagtcc gcgaagttgc ggcgctcacc cataccgacc 13560
agtgcggcgt ccgtgatgac ggaaccgact gcgttctccg cagcggcaga tgcaagcgat 13620
gcacccaccc ggccctcggc caccagcgca gcagaaccca cgcccagcac cttagcaccc 13680
gcagcggtga cgccgccgat ggcccagttg cccggatcgg tgaagcctgc cacgaggtcg 13740
tagccgaagc tcgaccagcc ggtctgctga tccttgatgc gcatggagct gcgacgcatg 13800
aactgccggt cgctcacgta cttcaactcg ttcggcgagt acgctgcatc gcggatgtct 13860
tccagttcgt cggcagtcat ccacggcttc tcccattcct tcgggtcgta cttccagccc 13920
tccgggatgt tctcgtcttc gcgtgcgagc tggttgctga tcgcggcacc gatgccgctg 13980
tcgaacgccg cgccccactt atcgaggaag gtcagagaat tgcgcagctc ctcgtccgca 14040
cgatcctgtg cgagtgcctg ctcggtctga tccagcagcg ccttgcgagc accggtgttc 14100
agcacatcca cttcctgacg cacccaccac ggcacaacct gatccgatgc acggacggcc 14160
tgcgcccggc ccacggcctg cgcggcagtg ccgccttcct gcgcaacctg ccccatcgac 14220
agctcggcga tacccgcacg aatcttgccg atgtgttcct tcgcagccga cgccagagcg 14280
ttgcggcctt cgatgggagc gccggtccac gcatccggga tggacgcacc ggaccacgtg 14340
cggtcgcgca tgttcgggtt accctgccat gcagcgttga ggtctgcctg cgtggtgctc 14400
aggctgcgcg gctgcgccga cacagcggga gccgaagcgc cgcccagcac gctgggtgca 14460
taggccgcgt tctcctgtcc ccagttcttc tggttggtgc cgccctgata gatgcgcagg 14520
gccttggtaa cgtcgccctt ggcaagctgc atgttctcgc gcatgatgtc ggcgtacagc 14580
tcgatgctgt cttggaagtt gcgcatgtcg tacttgcgac cgttgcgcgc ttcccacgtt 14640
gcctgcgtgg tgggcatgac ttggaagtcg ccctgtgcac ctgccttgct gcggatcaca 14700
ccgtcagcgt ccttggcggc gcgcccgttg ccggactcta ctcgccactg accgcgcacg 14760
atggactccg gcactcccgc ataagcggca gccttggcaa tctgctggtc ttgggtcagc 14820
cccgaaagat cgagtgccat tgtctatctc cttacagttt gggttggatg gaaatgctct 14880
gctgaatcgt accgtcgttc atgcgaacgt agccctgcgg taccggggcc tgcaactgac 14940
gctgctgctt cgtgtgcttc tcgtactcgg ccttgatgtc gcgcccgtac accggcacaa 15000
ccttccagcc cttctcgcca acagcgttga cgtacagcac cggctcgccg ttctcgctgt 15060
ccttcatgcg gaacacgtcg gccttcagtc cggtgctcgg ctcgatgttg acccgccgca 15120
gtgctgcgtc gatagccttc tcgatagccg gtcccgtttc cttcgtgtcc atgaagccca 15180
gccatgcacc cacgccgccg atcactcggc cttcgtcgtc gcgcccgttc tcccatgcgt 15240
acttgcccgc gtcttccaga ccgtaggcca acttggcgtg ggtagccgca gcctgcaagc 15300
cttccgcaga agtgtccagc agaccgccgg tgtcagtgac cgaccggacg atggcgcgag 15360
catacgcagc tcggccagag tcgcccagct tgttgccgaa catgccgagg tacttggggt 15420
tgagctttgc gatagcgtca gtaacacgag cctgagtagc ctcggattcc ttacggctgt 15480
tcgcgtcgaa ctgcgtggga tcgtcggtgg tgatttcgcc acgggccacc tcgtaggcaa 15540
gctcgaagtt cacgccgcca ttctccaatg cttgcagctt gttgaagaag gtgttgacgc 15600
tatctccgaa gtacagcgca gcggtggtcg cgccggtcag tcggccctgc acgggctgac 15660
cgttcgtgtc gatgcgtgcg gcggtggtct gcttcagttc cttccactgc tggtacagcg 15720
cagccgctgc cgtgttaggc tggtcgcgca gcacagcgcg tgccgtggtc tggaactggt 15780
cggccactcc cttcagtaca gcgccgcgac cggtgttggc attccacacc agctgcgaca 15840
tggcgacctt cgggttcttc gccatcgtgt cgttccatgc ctgcaccgtg ttggcctcga 15900
tcagctcctt gtccacgccg ggaatgttga tagtctgcgc gtaggtgccg cgcttgaacg 15960
cttcgatgcc accacggatg tcctcctcct gagcagccaa cttctgctgg tcagtcagtg 16020
cagccttgga ctcgccgcga tccgcagcgc cacggtagaa cgcggtgatc gccgacttgt 16080
acgactcgtc cgtctggtag cgcgatggcg acaggccatc gagcaacgac agctgcgcct 16140
gcgtcagtgc ctcggtagcc ttctcgtcca tgggacggtt ggtgccgagc atcgccgtgc 16200
gcctcgccag ttcgttgaag ccggtggacg ccgacgagtt cgcagcgtac tgcgccttca 16260
ccagctcctg ctgctgccac gccgtgcgct ccttcgtgtg caggtccatc agcgaacccg 16320
cgcgatccat gaaggtcttc tgcaacaccg tgtccgcgaa accgttgccg gtcatccggg 16380
cagtggcgag gtcgttgaac tccttcgcca tagcctcggg cggaagctga cgcaactccg 16440
gcatgcgccg cagcaggtcg ctctccattt cgttgacctg cgagagcgtg ttgtacatgc 16500
tcgcgccaat ctcgtagttg gtgtcgccga agattttctt gtaccacggc tcggtcgcag 16560
caatctcgtc catggtctta ccagcgcggg cggcaacgaa gccatcccac atctgctgct 16620
gcttcagctt ggcggtgttc ggcgctgcca gttcctgcaa gaactcaggc aggttgctgt 16680
ccggcatcgt cataccacca gtgctctcga accaatccga ccccgagcga actgggccga 16740
cctgattcac accagcgcct tgaatgctgg tgttgcccga cagtgcgggg gtggcgttga 16800
tgccaccctg caatgcggac tgcgtgttga aggtaaccgt gccgatgtca gccattacca 16860
tgcgcctcct gagttgcctc cgctgccgct atcgaaccag ccgaggccgg acgtagtggt 16920
ggagaagttg tcgtagttgt tgccgaagtt gccggtgacc ttcgccccgg tcacggactg 16980
cttgccggtg ccgtcgttgt acccgaaggt agtggacgcc cacgacttgc cgccgcgatc 17040
attggactgc tgccacgcct gcatcccgtt ctgtgctgcg ttgttgaagg actgcgacat 17100
accttggaag ttgccgttgt ccgcctgcca cgtaccgact gcgaagtctg ccacggcctc 17160
gcctgccatc ggcccgccga agtaggtagc cacggccacg ccaatcagct tgcccaagcg 17220
acgcttcatc gccttcggct cgatgtgctg cgagtagtcg aactggccga acgtgcggct 17280
catgtccatc ccgttccatg cacgcgacat cacctgcgcc gtctgcgacg caccgcgaga 17340
ggcgagcaac gataccgcgt tgtcctgtgt ctccttgtcc atttccgcct gcaagcgcac 17400
catcgtgtcc atcaggtctg cggacgaacc gccaacgccg acagcaccgg cctgcgcctg 17460
aagggcaccg cgagcgttcg ccagttggag ctgggagtta agcccagccc cttggcgcgc 17520
acgcagctcc tcggacagcg cctccatgtt ggagttgtac tccttcgcgg cggcgtccag 17580
ctgggcgttg ttcttcaggg accgcatgaa atcagcgaag ccggtcttgg cgacctcgct 17640
gcggttcgtg ttgtcggtct gcgcaacggc tgcacggtag ttcgcacggg caacgcgggt 17700
ggtgttgttc gcttgcagga tcgtattaac cgaacccatt agctaaccct ccgtacattg 17760
ttgaaagtct gacccgtcca gcttaggccg gtcacccgga gcgggagcca atccttgctg 17820
cgaatctcgt aggtgcactc acgcacttcg cgcccgacgc tgatgttcag ctgcccccgg 17880
tacaccggtt gccgcgcgat caggttgtcg gagtcgccga ggatgcggcc ctcgaagtcc 17940
gtggtggtca cgttgctgtt gcgggtggta accgcagcga gcatgcccgc cgtgtcctcc 18000
acgtccaccg tcaccttgtt cagcgacata cgaccatcga gcacggcctg accgttctgg 18060
tcccgaggga acggattggt cgggatcacc agcgcctcag acaccacgcc gtattccagc 18120
gcaccagcgt cgaggtcatc gaactggtta aggaagtccg acgcctgagc gtagggacaa 18180
cccagcagga agtacgggct agtcttattg acggcgatgc ttgcgctcgc ggtctgtacg 18240
cccgtaggtg taccaaaact cgcccagctg tcgaggcacg catgcgagtc cggctcgccc 18300
ttcgggtcga acacgtcgca cgccaccagc gacccgcgtg cagtgaacac gaggccgttc 18360
ttcttgtagc tgctgacgcc gatgatttcg cccagcgccg ggtggtactg gaacttcgac 18420
cacgagtcca cgatccgctg cccgttgttc ttgttgtcct cgtaccggta caggtagaag 18480
tcctgcgggt gttcgcgtgt gcggaacatc acgaggttcg gcgcggtgag tgcggtgatc 18540
tgcaatggcg tgcccgacag ccacgagtcc agctcgttgg acatttcgtc cgtcaccgtc 18600
tgctgaccgt tgagcgcacc gacgcgcatc tgatgcaggc tcgtaccttc gctgccgtac 18660
tgcgcgtaga agatgtagtt gcccgaggtc tgcgccttag cgtccaccga atccttgttc 18720
gcactgaacg ccttgataag tgggttcttc ggactgaaca ccgagtcgcc gctgatgccg 18780
tactgctgtc ggtcaccgaa gatcacgagg ttgccgttgt agagcgtggc gtgccggagc 18840
acgtcgccct cgctaccgtg cgcgaagatt tcgatggggt cgctgtcttc gatggtaacg 18900
gcagagccgc gccagaagtt caggtagtcc gccgagcgag acgcattgac gtagttctgc 18960
gcgcccacca ccagccggtc ttggaagacg gacagcatcg tgatcggctt gccgatgaac 19020
cacgggatcg gcgaggtcag accgtcgcct gcgacgcgcg caccgtaagc cggatgctcg 19080
ccgctgcccg gagccagaga cgtgaggccc gcaccgttgc gtgcgatgta gcccacaccg 19140
ttgtagatgt agagctgcga cacgaggttg tcgatggtgc actccacgcc gggcgcttcg 19200
taccagtcca ccgatccgta gcccgaagtc tccccgttct ccaactcggc gcgcaggtag 19260
tacgtctcgt ccgcacctgc accgctcggc cgcacgcgga tgatcttgcc atgccagtgg 19320
aacttgaccg cgtaggttgc gtcggtaatc tcctgccctg cggcgtagaa ggtcgtgccg 19380
tcgccctcgt catcaaccgt gatgtccttg aactggtcgt cgtcaatgat gatgccgccc 19440
ttgatgtacg tcgaggatac gcccgcgtcc accagcaaat ccgacagctt ctctgcgatg 19500
tactccgggc gcgtctgctc ggcgctggtc acgatccact cgttgactgc ggagttgtac 19560
tcgttcactc ggtcgttgac gtacttctga tacgccgggt ccggctccgt gccaccgggc 19620
aggtagaacg ggatgtcgct cgtgtcgagc gtgccggggt acgatgcggt caccgtcttg 19680
aacgtcaccg tcagcaccgt gttgtccgtg cgcgtcaggt tcaccgtgta cgtggtattg 19740
tacttgccgg tgcgaaccca tgcggcgagg cgctggatgt tcgtctgctc ctgccacacg 19800
ttggtgctgg tggccgaagg gatcgtggtg ttgcctgcga tgtagacgta gctgccgatg 19860
gccgcgagcg atgacgcacc gccgctgacc agcgtgttga cccacgtgct gttctcgtag 19920
accagcggga tgaacgcctc gtccgtcttg ttgtagagga acgcgaacgt cgcactgctg 19980
gacggcgaag cgtcgcggcg gtacagcagc gcgtactctt tgccctcctg catgaagtcg 20040
aacacgtcca tgttgcgcag ctcggccgag gtgacaacgg cttgggaagc agcgaaccgc 20100
gtaccacgac ggcgcacaag gccgttcacc ggatcggaca gcacgttgat ctgctcgtgc 20160
gtctggcccg gccgacgacg gtgcgccggt ttctgactga caccctgtag gatgtcggga 20220
taggttccgc ctgcctttgc catgatgtct ccttagccgg ggaaagtgta gtggctatgc 20280
cacgggtgtc cgctgtagtt gaagtaccgc tgacgaagaa cgtccacgga tgcgcgagcg 20340
aacagcgggt tcgccttgat ctgccggatg tgctcggcct tcaggttgat ccacgaatcg 20400
tcgcgctgct gcttgatctt ggcgtacttg gtgttgtcgc cgtcgaagtc agactggaag 20460
cgcagcacgc tgtcgtcccg cacgaacatc tgaacctcgt aggggaggtc atcgaagtcc 20520
agcagccgga ccaacttggc gtgcacgttg tggtcgaact cgtaggtgtt gcgcttgcgg 20580
tcgtacagtc ggcgtccgcg ctgcaccacg tggccgaatc ggctcgggtt gcagtccagc 20640
ttcagcacgt ccgttgggac catgatgtac ttgctcgttg cctgcggctt cagctccacc 20700
cattcggtgt tgaaccacag cgcgagcgac gacacgtcgc ggtgcacgcg gtcgatcaag 20760
tcaactgcgt cgtccttgta tacgtggtca tcttccagcg tgttgagctt cgtctcgccc 20820
atcgtcttga tgcaggcgtt cacgatgtcg agcttggtga tgaatgccat ggtgtctcct 20880
gagaggaaca aggcagagac gccggagcgc ccctgccggg atgttgctta gtccgcagcc 20940
agagccagcg aggccggtgc gtactccacg tcctgcgagc cgatttcgtc atcgacgaaa 21000
cggacacgca cgacccagcc cgaggcgatg gcgtcgtagt ggacgccgac gacggtgcca 21060
acggagttgg cgggcagacc ggccacgcca gttgcggcga cgacacggtt ggaaatctcg 21120
aacggtcggt caaagaccat gatggtgctc ctgtgttggt ggaaggcgag gcgcggtttg 21180
agttacccga aggattcagg gaccgcgccc agcggattag gcaaccttgt cgatgcggcc 21240
cgcgttctcg acgcgatcca gaccaacggc gaaggacatc cacgagtcca cgaagtagca 21300
cttcgaggca tcgctccacc acaccgagga ctgcaacggc agcgcttcgg cgatcatcag 21360
ggccttcggc gacacggcca cggcgaccag cgtgctgaag tcaccgtcgt agaagtcgtc 21420
gttgtcgtcg ttcgacagca ggtgaccttc gatcacggtg ttcggcaggt tctcgctcga 21480
cagcaccggg acgccccagc cgtggaaggt cgggatgccg gtcagcttgt tgccgtccga 21540
ggtcaggtac tcgccgttgc tgatctgctc ggcctgctgc aacaggtaga agatcgccgg 21600
acgcacgatc aggaccacgc catcacgctg cggcttgacc ttcttctcct cctccatgcg 21660
ggagaacagc ttgccgatgg cggcgtacac cttggccgga tcgttctcgt caccggcagc 21720
agccagaacc acctgcgtgg caccgaagtg gccttccggc gcgcggccgt tgcgggtgaa 21780
gatcgactgg gtggcggacg cggccttcgc ggcggcgatg ctgatggtca ggtccacgaa 21840
ctcggcgatt tcctgaccct gctcgatggc gatttccttg cgggtgtcga tgtcggtgaa 21900
gatttcgtcc agctgccaga tgctgtgacg cgacagcagc atggtatcga cggtcaccga 21960
cgccttgctg aagtccaccg gggtgccgtc cggcgcatca ccacgcttca gcttctggag 22020
cttggtgcgg ccgatggcct tcttggtcag ggtgttggtg cccttgacct gcttgcgcgg 22080
gaagaacttt tccagcaccg agctggacac gtaggtgtgc tcgacatcgc cggtgaactg 22140
ttcgaggatc agggcgtttt cgttgccgga ctggttgacc tgattcggca gaaccgggac 22200
ggtcggaaag atggtgctca tgctttatgt tgctccttgg ttcttagtgg atgacgcgcg 22260
cgttgagtgc acgcagttct tcggagtggg ccgcaccacg gatgcccttc tcggcaacca 22320
gcttgcggta ctcagcgcgg tactgctcct tcgtgagagg agcgacgccg ttgatcgacg 22380
cgaccggagc ggcggcattg gtagccggtg cgcccttgac ggtcgtcgcg ggattggcct 22440
gtgccagagc catcaggtaa cgcgcggtcg cctgtgcgcc gaagccacca gccgacagcg 22500
attcgcctgc ctgcttcagt tggtcggcgg gcaggttggc ccgtgcgaac tgctggatcg 22560
cagcccagtt ctctgcgcca cccacggctt cgtggacagc agcaacgttc gcggcgtgag 22620
ctgcggttgc agcagcggtg acacgagcga tgctttcctt cgccagagcg aggatcggtg 22680
cggaaccctt ggccttgtcg ccgaggactt ccagcttggc ttccagcagg gcgaagttgc 22740
ccttcgctgc ttcggtcagt tcgcggctgt cgatgtcgag gccaacggtg ttgacgaagt 22800
attcggccgc gatgttcagg ccgttgtcgc cgtagtccgg aatagacaca gcggtcggcg 22860
cagcgttcgc cggggttgtc gatctgctgc gatgcgttct gcatgaccag ctgctgctgt 22920
gcacgctgct gcatcatctg gccgtactcg gcctcagtca gcacgtagac gttcgggtcc 22980
aagccaccag cggcggcgag gtctgcgatc cacgcggaca gcttcagtcg ttcgagaatc 23040
tgcggcggca gtgcgaggac gttggcgagg ttgctgccga actccaacag ccggtcacgg 23100
tcacccgttc gggagagcgc ggccaagccg gtgatgatga caggctccaa gtccttgccc 23160
cggatgtcgc ggcccgtgag gttcatcacc caaaaggcga cgggcacctg cacgtccgca 23220
gcgatgcggg agtagccacc acccagcccg ccttccagtt cttcggcgac catgcggatt 23280
tcctgcgcgg tcacacgttc cgcctgacgg gtcaccgccg tctgcaacag gaagcctgcg 23340
ccgatgcggt tgatgtaggt ggactggatt gcttggttgg tttgcagcac gccatcgacg 23400
cctgcgttaa tcagctcgat gtcacccttc atgccgggga tggccgctcc gttgggcgat 23460
gccatgaagt cctcgacgga ggtctgccct gcggggttca ccagccaacg gaactcggat 23520
gcgaggatcg cgccctgcac tgcggcctcg gacagcatcg agagcgactg gaagtctgcc 23580
tcgtagtcct cgaccaagcc tgagccgtag tcgtcgcctg cggccaagtc ccaactcacc 23640
gcccggtagg gcagcttgtc tgcggtgtag gtcgaggaga agttggcggg cagcttgcag 23700
ttgcaaatcc aaacgtcttc gttgtacttt ccggtcttgt ggtccagctt gacccacttg 23760
tactcccaca ccatgccgtc gtcatcgggc ttgaagtccg ggtgtgcgac aatggcagcg 23820
agtgctgcgg gttccagctg ggtcttgtgc accttctccc gaaggatcag ttcgtagacc 23880
tcgccggttc gagtgcgctt gaccacgtag tgccgcatgt tgagcacgcg gatcgtatcc 23940
ttctccaaca ccatgagggc gttgcccaac acgatcagca ttttcagcag gtcgtacagt 24000
cgggatcgga tcgagcgctt gtcgagttcg agactcgcgt cctgctcggc cttcgcaagc 24060
atcgcctgca tggcctgcaa gtcagcgcct tgttcctgcg cggcctgctt cgccttgcgc 24120
ttcagctcca accggaagaa cgggcgggac ggtgcgaaca gcgacatcat cagcttgttc 24180
gccagatggt tcactgcctg cgcgccgaga gactggaacc cgtgggtcag cggctcggtg 24240
tcctgatcgc ggttcttgcg agggaacagc ttaggcacgg tccaactcga atagcgttcg 24300
agccggtaca tgatgccctg ccgcttgttg tcgtaagcct cgaactggcg gtgagcggtt 24360
tggatcgaat ggtccatgct cacctcacaa caggtctgac ccggccacgg tcgtcagccg 24420
ggaagctgcg gtcgtggtca cgggtcgggt gtaggcggag cgggcggtct tgcgtcggcc 24480
gatcaagtcg gcggtctgca agtcgaggtc ggaagtaccc agccgcacat cgacggtatc 24540
gacaggggca ctcagcaact tctcggcgta ctcggatgcg gcctgctgtg cagctgcctg 24600
tgccatctga tcggccatcg cctgaatctg gtagttcgtg gattgagttt ggaggttggt 24660
ctgcttgtcg atagccgccg cttgcttcag cgcagccttc ttcgcagacc caccaccaac 24720
gatgctaccc ataggttaac tccttcgctt ggtgaattgg ggagcatgcc gttccagcag 24780
gtagccgtag cgtgcataca tccgtgtgag tgctcgggag ttccgagcta gtgcaccgcc 24840
tgcgctcagt gagtcgcagt ggttgatgtc agccagatgt tccatcacgg ctatgtaccg 24900
gtcgaaggaa ctgcccggcc caattcgcag caccatgttc tcaacgaacg tagtacggtt 24960
cttggagtac cacggtgtga caatggcaaa gcatagcagg taagcttcat ctatgattac 25020
tgcatctact aagttctgag ttatcagagc tactgtataa tcaggatcat cacagttgta 25080
gcttacagaa gattctctgt tgtctacctt actatcttct ctagatgaca gatacaaagc 25140
ggccgttaaa gccgccttga tctgctcact atcacccgtc gttggtgttc ggtacggagg 25200
tatcaacggt gaaaccctcc tgtaagatgt tcaggacgcg ggccatcccc aactgcatac 25260
ctgcctccaa gggcgtggta ttcgagttga cggacagcga cagcccggac agtcgccgca 25320
cggcggcaag ctggttcggg tggagcgtca tcaggacttt cactttggcg tcttcggcca 25380
tggtgtctcc tctgtgctat aggtgtacca aaaggtccgg caccggttcc gcttacaatc 25440
gcggccgctc caaccggacc tcggttggcc tgtgtgctat aggtgtactg aaaccccgtc 25500
agctgaagaa gtacggagaa tcaaggactt ggcgcaggtc cagctcgccc atcggcggcg 25560
gctccggcag gaacgggtag gccgagtgga aggccgagag cacgtcgtgc cgctcgtaca 25620
tccccacgaa cacctcccgg atgatgcggt acagggccgc gctgtcggcg gcgtgggtac 25680
cgaagtcgtc gtggatcatg gcgaaggcgt cgatgccctc ggccttggcc gcgttgacca 25740
ccaacgtcag gtggctggcg tcgaggctgt gcacgaagtt cggggcgatg ccgttgcggt 25800
gccgggactt cttcaccgcg tcggtgctct tgcgcagcga cagcttggcg ttgccgcaca 25860
gcttggtgtt gatgcggtgc tcctcggatt cccagtacac ctgcaccacc gggaagccgg 25920
acggtgtgat ccaccggatg cggtcgtacc cttggttgag gatcgcaccc gagccgcgct 25980
gcaaccactc catcgcgccc gctgcggcga ccacggtgtc ggcgatggcc tcacccatgc 26040
gcttgctcag gaagcgcgct gcggggccgt acagggcctt gtcgagcgac gggaacttcc 26100
cgcacttcag gtagtcttcg atgatgaagt ccgcccacga ggactgccgg gagccgtagg 26160
gcagggtcat cacgctgcgc ttcaccagtg agcgtgtgag gccgtgttcg agccacagcg 26220
tgcggtaccc ttcggcgtcc tcctcctcgc ccagcaggat cgcagtcacg cggtctgcca 26280
ccatctggta gatgtcattc ggcagcggtg ccgggatcag gttcgtcgcc accccgcctg 26340
ctgcgtcgcg cagcatcgcc gagaagttct gaagcccatt gcacgaacca tccatgccga 26400
ctgcgatgcg gctcaggaag cgatccccga atacttgcca ctgctcgtac tccatgcacc 26460
agctcagaaa ctgcaacggc ttgtcggccg tcttccactc gtcgttgttc accgggtccg 26520
cagcgaaggc catgatctgg tcgtgatgct cgcgcaccca cttcacgcgg tccggcagcg 26580
acgccttgtc gtagccccat cggtttgctc ctgtgatgca gaaccaatcc ttcgcatcct 26640
tcgtcagcag cggcttgcct tcggcgaact ccaagagcgc cttctgcaag tccgaaccct 26700
gcggcgatac gccggtggtc tgcacgtact tgcggccacg gaagtctgcg aagtacacga 26760
agtagatgct cgggtactcg gcgaacttgc gcgccacctt cattgcgttg tagaagcggt 26820
tgcccttgtt ctggcgcagg cgctcgtcgg tgtgccaatt cgccatgtcg cgcttccact 26880
gcttgaactc caactcctga ttgtgggaca tggtttcctt ggtcatgtcc ttcgtcagcc 26940
actccggctt gcgcggcttc ggcagctcgc cttgcgaaat gatttcctcc atgtcgtgca 27000
ccttggcgat agccgacacg gcttccatca ggcgcttgtt gatacgccac ggcacgcgct 27060
gcaatgcgtt gatcgcggac atctcgttgc gcagctcggc agcgcggtac tcgtcccgag 27120
tctgagcgta ggtcttcacc atccacggat tgagtcggcg catttccttc gtgtggaatc 27180
caccgttgtc gatgtccacc cagtccatcg gcggctcgac acacggcaga tagaacggcg 27240
tggcctcgat cacgaagtcg ctgatctgcg taatcagctt gcgcacctcg tcggtcagca 27300
cgatcttgac gtgcgtcttg gtctggaact tacggtacgc tgcaccggca ctggccgcag 27360
tctccgtgac cacgccaatc tccaacatgc ccagctgtgc aagctggtcg cacaggtatg 27420
cgccgacctg atcgcgctgc gcctgccccc actccacgaa ctcgatgccg ttcttgcggc 27480
cctgcatctt catcaccgtc atgcggtggt tctcgttgac agagagcttg cggtccatgt 27540
cgttcatcag gtggtagaac agagtcggct ctgcgtcggc gaactgagtc agcaggtact 27600
cgtggtacac gttgatgcct acgttcttca gcacgtcgcg gtcgctgcct tcgccgtgct 27660
tgtcacgcat gcaggaggta agggcaccgc gaacggcgac gaaggccaca gcctcgtacc 27720
actcgcgggc ttgttcctcg gcgctcacgg cctgcccacg cttgttctgg cgctcgggat 27780
cgtacttggc gcgcagaagt ggcacgtgcg cttgggcacg gccgatgcgc ggggagtcga 27840
tgtccgcacg gatgcgctcc gcaagcggaa gcacgaaccg gcggtacact gcttgggcgt 27900
aggggttgtt gtccgcgccg cccttctcct cgttgcgggc gatggacttc tccatgcgct 27960
ccttaccgaa ggcgtaggtt tccagttcga gttccacttg cgagagcatg ctacctccgt 28020
tgcttgatgg tgaagtccgt cggcgggtag cgcttcagga tcatgcagac cagtgcggtg 28080
tgcatttcgt tgatgatgtg cggccacgca ggaccgccga cctcgtagct cgggatgcac 28140
tggcggttga agaactgctc ggtgccgcac cacgcggtgg cagtcacggt gacgtggcct 28200
ccaccgtagg gagagaagcg gtcaccgtcg aaatccagct cgggctttcc gaatcgaatg 28260
tccatcagcc cctccgctcg gcgaggaagc ggtccagcga ttcgatcacc gcatcgcgca 28320
ctgtctcgtt cggccacagg gaaccggcgt aatcacggaa cgggatgctc actcgcacct 28380
ccaccgtgtc gcgctccatg ttgtgccaca cttcggtctg aacgttgctc accggtcggc 28440
ctccttcttg tcatggcgga tgcccttgta gcgcggctcg cgcagcaggc cgtacttgct 28500
gaaggacatg gcttcgattt ccacgatgtc gccgacgacg ggaacttgcg aaagctggtg 28560
cggcacgccg ctcccgacct cctgctcctt gccgtcgccg aggtccacga acacggtgta 28620
gacatcgcgt ccggtcttct cgccgacagc gcggccgatg cgcagcacac gcaggtccag 28680
cgtcagggta ggcttgacct tgacgacttc gccgttcgtg cccttgtcgc ccttcaccca 28740
cttgccgctc ggcttgcgca ggatcaggcc gtcgtatgcg ccgccctcgc acagcagctt 28800
cgccgcctcc atgctgccca gctcttggtc gatcaggaaa ccctgcgact cggcgtagaa 28860
gacaggcgcg gtgccttcgg cgatggcatg acccatgctc ggcaggcggc tcacccgctc 28920
ctgatacacg aggtcgctgt agccctgctc ccattcttcc agcgtcacga agtcgaacac 28980
cacgaacatc ggctcggtgt actgcgtgcc gtccttcttg cggaacatgc cgctgacggt 29040
gggctggtcg atgtgcgggt gccagtattc cccgaagtac acgccgaccg ggaagaacgg 29100
agcggaggcg agggcattct cgatgtggtg cgcgctgacc acctgctcgc cggtgcggga 29160
gaacaggtcc accacaccgc gatccaccgg ctgcatctgc acgtcgccga ccttgatggc 29220
atgcaggccc ttgttcacgg tgggcaccca gtggccgcgc ttgatggcga ccatgttgca 29280
cccgtcgtac ttcggctgtg cgatgtactc ggtgcgcagc tgctcctcgg tgggccggag 29340
cttcttcttg atcttgtcct gctcgactgc cttgtggacg atgtagctca tgcgtggtcc 29400
ctccaaatct ggaacatggt gaccggcacc ttgtaccacg ggacatactg ctcccagtag 29460
gtggccttcc cgtcgtacca ctgcacgcgg aagttgccct tggtgaaggc gcggttgatc 29520
ttgaacccgc gcttgcgctt cttgtatccg ggagcaaccc ggacgttatc ccgcttgatg 29580
tcaaacatcg gcgctgtcct ccttcttgct gccgtcgcca agcgcagcgt tgaagcgtgc 29640
gatgcgggac gacatgaact ggttgacgat ggtgccgatc cacagcttgc gcggagcgag 29700
cgaaccacgg tagccttccc acgcctcggc gatacgggtg tacagctccg cctgatcttc 29760
ttccggcagg ttcgggaacc ctgccttgac cgcgttctgc aacgcctcct cgtggtcggc 29820
gctctcgccc cacgcaacgg tgttctcgta ggacttgatg agcatggcgg tctggttctt 29880
gtagagcatt cagttctcct tcgacgggaa tggaacgaaa cctgcgcggc ccttcatcac 29940
taggatgcag aaggcgtcgt cagggtaggt acgatgccag agtgagttag cccactcgac 30000
atcgaactgc ttgcgggtac ggtgctcctc taggaagcgc ccactcggcc tcatgtggga 30060
cgtaccgatg cacttggccg cagcggacgc aggccatcag tcacccttgc tgaaccagcg 30120
gcggatgatg tactggcgca cgaagctgat gatcgtgaag gcaacgccga tggcgtgcgc 30180
cgacatgagc gggtgctcgg gatgccacag cagcggaagc aacgcgatgt tcgcgcagta 30240
gttgatgctg tagccgatgg cgatgttgac ccacgcctcc ttgatcgagc gccccttact 30300
ctgcgccacg ggccttctcc tcctcgcgca gttccagctc caacaggaac agctcgttga 30360
tgatgtggtg tgcgcggtgg tggatgcccg tctccgggtc cagcttctcg ccgaggcagc 30420
gcttgttgtc gtggcggtag ctcgcatcct gatagcgttc gagcgccttc tcgacatgct 30480
gccacgagtg cgccttgtac ttcttcgcac cgaaggtcag agtgtcgatg acgcgggcca 30540
gtgcacgcgg cacaccctcg tgcagcagcc gggcctgcgg cttgccgccg tcatctttca 30600
ggccgacgcc acggtcggac atcgtgcgct cgtcttcgtt ccggtagttc agttgttcgg 30660
gcaagggaat ctccctcctt gtggtggtga cacctcccac gcccatcagg cgggtgagga 30720
agtgcgggcg gatgatggac tccttcagct gtccgtcgaa caggactccc acgtcaacgg 30780
cactgatcga gcgaaccacg ccggacctac cgacgaggcc cggagcggag ccggtgtagc 30840
gtacacggtc gccgtggcga acgtcggtaa gtctcatgcg tttctcctta tgcgggtacg 30900
gtgatgtgct tgcgcgcttc ggcactggtc gtgagcttgc gcccacggct gtagccatcg 30960
cacacggtgc actgaagcag gaggtacgtg ccgacctgtg tccgggccgg atcatcctgc 31020
gggatcattt cgttgcagcc gcacttgttg cagcggtgct cgctgccgtc ggccttgtag 31080
tacacggcga cgttcgggtg ctgcgagtac cagccgcgca gcttcttgta ctccaacttc 31140
atggacttca cgtcgatgcg gttgtacgct tcacactcgt cccatgcacc gggcaggttc 31200
tgcatgcagg ccacccacag gtcgaagccg gggaagttgt ggtggtcgta cttgcgcagc 31260
tccggcacga tcacgccggt ggtgtactcc aacttctgcg aggtgaaggc gaactcggtg 31320
cggttcagca gcatcgggtc gatcacctgc acgggtgaca caggaggcag gccgaggatg 31380
gcgaggcgcg ccttgatctt gcgcagatcg aacttcttgc cgttgcgagc gagcacgaag 31440
tcagcagcgt tgagcaggtg ccacagcgcc ttgccctgtt tgcggtcatc gaacacgtca 31500
cgcacgttgc gctggtccat gtagaacgac tcctcgtcgt ccagccattc gcaagcgaac 31560
gacatcagcg accagtcacg cttgatctgc ttgacgctga agttgttgtt gaacatcccc 31620
cagccataga actcgatggg gaacgtctcg atatcgaggc agagaatctt cgggccgcgc 31680
ttcggtgctc ggcgaatgaa ctgggtaagg gtcacggtgt ctccttgttg agtgaccgtc 31740
gcttacgcgc cttcgcattg cgagcgatgc gcttctcgtc gtcggtcttg taggtaggat 31800
gcaggtaccc cgtgatgttg gtgaggtggg accgcaggta gttggggatg cccgcacaga 31860
atgcgaacac atcgcgcacg ccataccggc ctgcgttgtt ctcgaccttg ccgagcagcg 31920
agttgcatcc gcgatggagc accccgcgca cagcgccggt cttgtggtca tggtccagaa 31980
caggatcgag cggagctttc aggccgaact gtccctgaca cagacagcac cggttccctt 32040
gctccgctgc gagcttgatg cgcaacagct tgacttgcgt tgcagtgagt cgcttcatgc 32100
tttgtatgcc tccaagattg cgcgcttctc gttcacttcc tcggcgaggt cgtagaatgc 32160
ttgcttcagc tgcggcccga acacgtcgag cttcagcacg tccagcagtt cagcgtcacg 32220
atccgtccgc atccacagaa gcacggcctg ttcggcgaaa gtcttgcgcc agtcggcacc 32280
gagcacctcg ctgtacttgc cgctcaccag cttcaccgct tcgaggttgt ccttcgtgtc 32340
gagaagcagc tgctccgctg tcaccttgcc caccttcggc agaccgggta tgttgtcggc 32400
ggtgtcgccg tggagcatct gcatccagaa ccacttgtga ccgtactgca atccgttctc 32460
gcccacgatg tcgtaagcgc cgagcggaac ctcggtgatg tcgtagccat cccagtctac 32520
gtgcacgccg cagaacattc gcatgtcctt gtcggcgctg tgcaccacgt gcagtatgtt 32580
gtggtgcaat gctgcaccct cgttgacgta tgcgatgccg tcgtcggcct cacgtgtcag 32640
ccagattttg ggagtgaaag cgtcgccctc gtatgtctcc atccactcac gaaggaacgc 32700
ccagttgttg ggcttgcggc ctgcttggcg ctgaccctga tacggctgcg tggtagcagc 32760
gaggaagcga tcacccttgg tagacgcggc gtgcgtcagg tgcatcacga tcttgcccgc 32820
tccgctgatg tgcttcagct gtgcgcagcg ggagagcacg ttgcgtcgtg catcccccgc 32880
cgaacagttc tcgctaccag ccgcgaagta cgccatgtag tccccgtcaa cgtgagcgat 32940
cacgcgcggg ttgtgctgac gtaccacttg caccggcatc ggggagcgct caccagccgc 33000
agccatgaga gcgctcaggt ccatcaggcg atgcccgcca tcgggtcgct accatccacc 33060
gggaacggcg gcttgtcgtc ggccggagca gcatcggtgg gcgcggcgtt cgccacgtcg 33120
ccgaccgctt cgtccagagc atcgacgcct tccttggtga ccgtgctgct tcccgccaac 33180
ttggcggcgg cgtagtcgta gcacggcagg cccttgaagt tcagcgccgc agcgatgcgc 33240
agctgcgtct tgttcttcga ctgcgcctcg tgggtgacct tgccggtctt ctcgtccttg 33300
cgttccgggt actcgccggg gatgtagatg ctgtcccaca tgtcggcgtc ggcgaagtcc 33360
cacacgaacg cacccagcgg ggtgatctgc tgcgggaccg ggatcacggt gtccacgaac 33420
ttgccagtcg ggatgccgtt ctccatttcc gggaccggca tgaccggctt gcggatggaa 33480
gccttgtcga tttcggcgaa ggtcttgttc gggtcgcgct tgcccggctt gtggatgact 33540
tccagcagca caccctgacc cagcagctgc acgaagtggc gctcgtcggt gcgcaggcgg 33600
ctgaagattt tgaagtagtg cgccttctcg ttggtggaca ggcccagcgt cagcgtcatg 33660
cgcaccggaa ccggaccatt gtccgtctcg atgtgcgggt gctgcttgcc ggtcagctcg 33720
aacacgatgg cgacttcatc ctgcgtcttc ttcttgccct cgaactcgaa ctcgtgcttg 33780
cccagctcgt agtacccgac gatgcgggcg gtggtcacac cggcagcggg cggggtgtag 33840
tcgaagttgc caccactggc gaccgactgg tccttgccct gcgtggcgac cttgttcaac 33900
agtgcgtcaa gactcatgcg tgtggttctc cttattcaaa cgacgggttg tgattaccta 33960
cgaagtcctt gcggatcgac gctcggtact cggtgaccag accggcgaac tctgcgggag 34020
ggttcttctc ctccatcatg ttgtcgccca tcttggtttc gcacgggacg ccaagcggca 34080
gcgtccagtt gaaccaccac tccatataca ccgacgcttc cagcatcgag gcgtgcagca 34140
gagcagcggc cttggttgcg atgtcgccgt ggctgtccac gtacacggca tcgtgcacct 34200
gattcacgag caacgcacgg cccaaccatt ccttcggttc ggtcacgtac ttgcggtaga 34260
aagcgcgcag gctgacgtac attgcagcct tggcccactc accaccggtg ccctgcatcg 34320
ggtagttctt gatttccgtg ggcgagaacg actgcgcgca gccgcccttg ctcgcgggct 34380
tgttggcgat gaacaccgga gacggcgact cgctgaagga atacatcttg ccatccggcg 34440
tggtccagtg cgaccggccc agctgacagg tcagcccggc aggcacgccc tgacgacctg 34500
cgtccgggtg cggcacgaag cggttggtgc tcacgcggtt gcgcttgatg gcctcgacca 34560
tgcggtcgat gtacgcaccc agctccgggt aacgctcggc ctcaccacgc accagcgcct 34620
cgacttcctc ctccgacatg ccttgagttc ggccgtcgct gttgcaagct cgacctccaa 34680
ctccttcgcc ttcgca 34696
Claims (7)
1. A cross-species cleavable Xanthomonas phage is characterized in that the Xanthomonas phage is Xanthomonas phage Xanthomonasphage) GJ19P1 with a preservation number of cctccc NO: m2020805.
2. A composition comprising a cross-species cleavable xanthomonas phage of claim 1, wherein: the composition at least contains a xanthomonas phageXanthomonas phage）GJ19P1。
3. The composition of claim 2, wherein: the composition includes a chemical sterilant.
4. A composition according to claim 3, characterized in that: the chemical bactericide is amikacin 700 times liquid.
5. The composition of claim 4, wherein: the composition comprises xanthomonas carpet grass bacteriophageXanthomonas axonopodisphage) YHC5 with preservation number of CCTCC NO: M2018579; pseudomonas syringae kiwi fruit pathogenic variety phagePseudomonas syring pv.Actinidiaephage) PSA-P1 with preservation number of CCTCC NO: M2020252; lei's bacteriophage of SolanaceaeRalstonia solanacearumphage) GP3 with a preservation number of CCTCC NO: M2016635.
6. A kit comprising the composition of claim 5, wherein: the kit is provided with the xanthomonas phageXanthomonasphage) GJ19P1 or phage containing XanthomonasXanthomonasphage) GJ19P 1.
7. Use of a composition according to any one of claims 2 to 5 for controlling bacterial diseases of plants caused by xanthomonas, characterized in that: wherein the xanthomonas phage Xanthomonasphage) GJ19P1 as an active ingredient of a biological disinfectant or a biological pesticide.
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|---|---|---|---|
| CN202110243645.8A CN113151192B (en) | 2021-03-05 | 2021-03-05 | Xanthomonas phage capable of cross-species lysis, composition, kit and application thereof |
| KR1020227008496A KR20220041222A (en) | 2021-03-05 | 2021-03-18 | Santomonas phage capable of interspecies lysis and combinations, kits and applications thereof |
| PCT/CN2021/081420 WO2022183531A1 (en) | 2021-03-05 | 2021-03-18 | Cross-species cleavable xanthomonas phage and composition, kit and use thereof |
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| CN202110243645.8A CN113151192B (en) | 2021-03-05 | 2021-03-05 | Xanthomonas phage capable of cross-species lysis, composition, kit and application thereof |
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| CN113151192B true CN113151192B (en) | 2023-11-24 |
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| KR (1) | KR20220041222A (en) |
| CN (1) | CN113151192B (en) |
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| CN108676778B (en) * | 2018-04-20 | 2022-09-20 | 南京农业大学 | Bacteriophage for preventing and treating soil-borne bacterial wilt and application thereof |
| CN114934025B (en) * | 2022-06-22 | 2024-05-28 | 北京林业大学 | Pseudomonas syringae phage CXP1 and application thereof |
| CZ2022395A3 (en) * | 2022-09-20 | 2024-03-06 | Biologické centrum AV ČR, v. v. i. | The Xe_NED111 bacteriophage strain, a preparation based on this bacteriophage strain and their use against bacterial spot on tomatoes and peppers |
| CZ2022397A3 (en) * | 2022-09-20 | 2024-03-06 | Biologické centrum AV ČR, v. v. i. | The Xe_PB119 bacteriophage strain, a preparation based on this bacteriophage strain and their use against bacterial spot on tomatoes and peppers |
| CN116926017B (en) * | 2022-12-05 | 2024-10-22 | 菲吉乐科(南京)生物科技有限公司 | Xanthomonas phage applied to bacterial diseases of feed crops |
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- 2021-03-05 CN CN202110243645.8A patent/CN113151192B/en active Active
- 2021-03-18 KR KR1020227008496A patent/KR20220041222A/en not_active Application Discontinuation
- 2021-03-18 WO PCT/CN2021/081420 patent/WO2022183531A1/en active Application Filing
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| CN113151192A (en) | 2021-07-23 |
| WO2022183531A1 (en) | 2022-09-09 |
| KR20220041222A (en) | 2022-03-31 |
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