Xanthomonas campestris pv. citri bacteriophage and uses thereof

Abstract

The application discloses a citrus subsp. Citrus bacterium of xanthomonas campestris and application thereof, and relates to the technical field of biotechnology. The citrus subsp. Citrus bacterium of xanthomonas campestris is preserved in the China Center for Type Culture Collection on July 4, 2024, the address is Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: M 20241486. The citrus subsp. Citrus bacterium of xanthomonas campestris can specifically inactivate the citrus subsp. Citrus bacterium of xanthomonas campestris, can complete mass proliferation only by using a small amount of initial bacteriophage, and can provide a high-quality bacteriophage strain source for industrial production of bacteriophage bactericides; and a person skilled in the art can prepare the citrus subsp. Citrus bacterium of xanthomonas campestris provided by the application into various products applied to detection, disinfection and plant protection and the like according to the description of the application and common knowledge in the field, and the products can be applied in industry.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a Xanthomonas citri subspecies citri bacteriophage and application thereof. Background Art

[0002] Xanthomonas citri subsp. citri is a Gram-negative bacterium of the genus Xanthomonas. Its short rod-shaped body is blunt-ended, measuring 0.5-0.7 x 1.5-2.0 μm. It is singly arranged, with a single polar flagellum. It does not produce spores, possesses a capsule, is oxidase-negative, and is catalase-positive. Xanthomonas citri subsp. citri appears as smooth, yellow, circular colonies with neat, sticky, and slightly raised edges on NA medium. The optimal growth temperature for this bacterium is 20-30°C, with a minimum of 5-10°C and a maximum of 35°C. Its lethal temperature is 55°C for 10 minutes, and its optimal pH is 6.6.

[0003] Xanthomonas citri subspecies citrus is the pathogen that causes citrus canker. Citrus canker occurs in all citrus-producing areas in my country, primarily affecting leaves, fruit, and branches. It forms corky, crater-like lesions with raised, double-sided ridges. Later, it can cause leaf and fruit drop and branch dieback, severely reducing citrus yield and quality. Once infected, citrus trees carry the infection for life. Citrus canker affects over 10 million mu (approximately 166 acres) of citrus trees annually, causing direct economic losses exceeding 500 million yuan. Currently, the prevention and control of citrus canker primarily relies on chemical pesticides, copper preparations, and antibiotics. Improper use of copper preparations can easily cause phytotoxicity and lead to outbreaks of spider mites. Long-term use of antibiotics can easily lead to drug resistance in pathogens. Furthermore, chemical pesticides are non-selective, killing both pathogens and beneficial microorganisms. Long-term, high-volume application of chemical pesticides also leads to the accumulation of pesticide residues in plants, impacting the quality of agricultural products.

[0004] Bacteriophages are a type of virus that infects bacteria. Their main chemical components are proteins and nucleic acids. They are widely present in soil, air, water, and organisms and have strong specificity. Virulent phages can adsorb, invade, replicate, assemble, and lyse cells in a short period of time, killing bacteria. Since Frederik Tword first discovered bacteriophages in 1915, a growing number of studies have demonstrated that phages have high antimicrobial activity and specificity, and are non-infectious to humans, other mammals, plants, and non-target microorganisms. However, current research on phages has mostly focused on screening and exploring their biological properties, with few reports on their application. Therefore, screening and applying suitable virulent phages is an effective approach to developing new antimicrobial agents.

[0005] Currently, research on Xanthomonas citri phages is almost nonexistent, and there are few reports on the use of phage therapy to control citrus canker caused by Xanthomonas citri phages. Therefore, the development of Xanthomonas citri phages for the prevention and treatment of related pathogens is of great significance. Summary of the Invention

[0006] The present invention aims to provide a Xanthomonas citri subsp. citri phage and its use to address the problems of the prior art. The Xanthomonas citri subsp. citri phage can specifically inactivate Xanthomonas citri subsp. citri and can be multiplied in large quantities with only a small amount of initial phage, providing a source of high-quality phage strains for the industrial production of phage fungicides.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a Xanthomonas citri subspecies citri phage (Xanthomonas phage) XP1, which was deposited in the China Center for Type Culture Collection on July 4, 2024, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO: M 20241486.

[0009] The present invention also provides use of the Xanthomonas citri subsp. citri phage XP1 in the preparation of pesticides or fertilizers for preventing and treating citrus canker, wherein the citrus canker is caused by Xanthomonas citri subsp. citri phage.

[0010] The present invention also provides a pesticide or fertilizer for preventing and treating citrus canker, wherein the active ingredient includes the above-mentioned Xanthomonas citri subsp. citri phage XP1.

[0011] Furthermore, the pesticide or fertilizer also includes pesticide or fertilizer acceptable adjuvants.

[0012] The present invention also provides use of the Xanthomonas citri subspecies citri bacteriophage XP1 in preparing a disinfectant for Xanthomonas citri subspecies citri.

[0013] The present invention also provides a disinfectant of Xanthomonas citri subsp. citri, wherein the active ingredient includes the Xanthomonas citri subsp. citri phage XP1.

[0014] Furthermore, the disinfectant is a liquid preparation.

[0015] Furthermore, the disinfectant includes auxiliary materials; the auxiliary materials include aqueous carriers.

[0016] Furthermore, the aqueous carrier is SM buffer, phosphate buffer, NB culture medium, LB culture medium or chloride-free water.

[0017] The present invention also provides use of the Xanthomonas citri subspecies citri phage XP1 in preparing a detection product for Xanthomonas citri subspecies citri phage.

[0018] The present invention discloses the following technical effects:

[0019] (1) Xanthomonas citri subsp. citri phage XP1 is a virulent phage isolated from nature. It does not contain virulence genes or adverse genes, its DNA cannot encode proteins that may cause potential health risks, and it does not have the possibility of carrying lysogenic genes. The present invention does not perform any genetic modification on the test phage.

[0020] (2) Xanthomonas citri subsp. citri phage XP1 has a high fermentation titer. During fermentation, the optimal MOI for infecting the host Xanthomonas citri subsp. citri is 0.01. It is a virulent phage with high affinity and lytic ability. At the optimal MOI, the titer can reach 1.13×10 within 12 h. 9 PFU / mL or higher. Xanthomonas citri subsp. citri phage XP1 can specifically inactivate Xanthomonas citri subsp. citri phage, requiring only a small amount of initial phage to achieve large-scale proliferation, providing a high-quality phage strain source for the industrial production of phage fungicides. Those skilled in the art can, based on the disclosure of the present invention and common knowledge in the art, prepare the Xanthomonas citri subsp. citri phage XP1 provided by the present invention into various products for use in detection, disinfection, and plant protection, and apply them industrially.

[0021] (3) The Xanthomonas citri subsp. citri phage XP1 of the present invention is a strictly virulent phage with high specificity and lytic properties for host bacteria and a wide host range. The lysis rate of typical Xanthomonas citri subsp. citri from eight different counties and districts is as high as 100%. Xanthomonas citri subsp. citri phage XP1 can be used as an active ingredient in various products for environmental disinfection, including but not limited to liquid immersion, spraying, and combined use with aqueous carriers to disinfect water distribution systems, irrigation facilities, public and private facilities, or other environmental surfaces, and can effectively control the growth and activity of target bacteria. Liquid immersion and spraying forms include but are not limited to detergents, disinfectants, detergents, etc.; aqueous carriers include but are not limited to SM buffer, phosphate buffer, NB culture medium, LB culture medium, chlorine-free water, etc.

[0022] (4) The interaction between Xanthomonas citri subsp. citri phage XP1 and non-host pathogenic bacteria was not able to identify any of the six tested non-host pathogenic bacteria, indicating good specificity.

[0023] (5) Xanthomonas citri subsp. citri phage XP1 has good stability. At -80 to 28°C, the titer can be reduced by no more than one order of magnitude within 24 hours. At pH = 5 to 11, it has good stability and the titer can be reduced by no more than one order of magnitude within 2 hours.

[0024] (6) Xanthomonas citri subsp. citri phage XP1 can be used to prepare compositions, reagents, or kits, and can be applied to the rapid detection of Xanthomonas citri subsp. citri, including but not limited to detecting Xanthomonas citri subsp. citri in target samples in the form of test strips, kits, etc., or screening target pathogens in samples, which can effectively ensure the sensitivity of the detection.

[0025] (8) The Xanthomonas citri subsp. citri phage XP1 provided by the present invention can be prepared by a person skilled in the art according to the description of the present invention and common knowledge in the art into a biological agent that can be used to prevent and control diseases caused by Xanthomonas citri subsp. citri phage XP1. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0027] Figure 1 This is the plaque image of Xanthomonas citri subsp. citri phage XP1;

[0028] Figure 2 This is an electron microscopic observation of Xanthomonas citri subsp. citri phage XP1;

[0029] Figure 3 This is a diagram showing the results of nucleic acid property determination of Xanthomonas citri subsp. citri phage XP1; wherein, M is Marker5000, 1 is DNAse treatment, 2 is RNAse treatment, and 3 is ddH2O treatment. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] The culture medium formula of the present invention is as follows:

[0036] The formula of NB liquid medium is: peptone 10g, beef extract 3.5g, sodium chloride 5g, distilled water 1000mL;

[0037] The formula of NA solid medium is: peptone 10g, beef extract 3.5g, sodium chloride 5g, agar 18g, distilled water 1000mL;

[0038] The formula of NA semisolid agar medium is: peptone 10g, beef extract 3.5g, sodium chloride 5g, agar 8g, distilled water 1000mL.

[0039] Example 1 Isolation, purification, identification and preservation of Xanthomonas citri subsp. citri phage XP1

[0040] The source sample of Xanthomonas citri subsp. citri phage XP1 in the present invention was collected from the peel of an orange orchard in Yizhang County, Chenzhou City, Hunan Province. The peel was homogenized with water and filtered through double-layer filter paper. The filtrate was centrifuged at low speed and room temperature, and then the supernatant was filtered through a 0.22μm filter membrane.

[0041] 1. Isolation of bacteriophage

[0042] (1) Add 1 mL of the filtered sample to 50 mL of NB medium and 1 mL of the host bacterial culture in the logarithmic phase. Incubate the culture at 200 rpm and 28°C for 12 h.

[0043] (2) Centrifuge the culture at 12,000 rpm for 10 min, filter the supernatant with a 0.22 μm filter membrane, and store at 4°C until use.

[0044] (3) Take 0.5 mL of logarithmic-phase host bacterial suspension, add 5 mL of semi-solid NA medium at no higher than 45°C, mix well, and pour onto an NA plate to prepare a double-layer plate containing the host bacteria. Take 5 μL of the filtered supernatant and spot-drip it onto the solidified double-layer plate. After air-drying under sterile conditions, place it at 28°C for 12 h and observe the growth of phage spot plaques.

[0045] 2. Phage Purification

[0046] (1) Puncture a single plaque with a sterile needle into 2 mL of logarithmic phase host bacterial culture, incubate at 200 rpm and 28°C for 12 h, filter through a 0.22 μm filter membrane, and perform a 10-fold serial dilution of the filtrate.

[0047] (2) Add 5 mL of semi-solid NA medium at no higher than 45°C to 0.5 mL of logarithmic-phase host bacterial suspension, mix thoroughly, and immediately pour onto a NA plate. Wait for it to solidify and set aside.

[0048] (3) Take 5 μL of each of the above gradient dilutions and spot them onto a double-layer plate. After air-drying under sterile conditions, place it in a 28°C incubator for 12 hours and observe it. A double-layer plate containing a single phage plaque is obtained.

[0049] (4) Puncture a single plaque into 2 mL of logarithmic phase host bacterial solution and purify at least three times as described above until plaques of uniform morphology and size are formed on the plaque-forming plate.

[0050] (5) Single plaques of uniform size and shape were placed in 2 mL of logarithmic host bacterial culture medium and cultured at 200 rpm for 12 h at 28 °C. The mixed culture was then added to 50 mL of NB medium and cultured at 200 rpm for 12 h at 28 °C.

[0051] (6) The mixed culture was centrifuged at 12,000 rpm for 10 minutes, and the supernatant was filtered through a 0.22 μm filter membrane to obtain the purified phage solution, namely, Xanthomonas citri subsp. citri phage XP1. Xanthomonas citri subsp. citri phage XP1 produces a single circular plaque on the Xanthomonas citri subsp. citri lawn, with a clear center and a diameter of 4-5 mm (see Figure 1 ).

[0052] 3. Electron microscopic observation of bacteriophages

[0053] The purified Xanthomonas citri subsp. citri phage XP1 solution was taken for electron microscopy observation: 20 μL of the sample was dropped on a copper grid, and it was allowed to naturally precipitate for 15 minutes. The excess liquid was absorbed from the side with filter paper, and 1 drop of 2% phosphotungstic acid (PTA) was added to the copper grid. The grid was stained for 10 minutes, and the dye was absorbed from the side with filter paper. After drying, the grid was observed by transmission electron microscopy.

[0054] The results are as follows Figure 2 As shown, under an electron microscope, it appears as a polyhedral, three-dimensionally symmetrical head and an inextensible long tail (see Figure 2 Based on its unique size and morphology, the phage is systematically classified as a member of the Siphoviridae family according to the International Committee on Taxonomy of Viruses (ICTV).

[0055] 4. Extraction and Sequencing of Phage Genomes

[0056] 200 μL of the purified Xanthomonas citri phage XP1 solution was taken and the phage nucleic acid was extracted using the FastPureViral DNA / RNA Mini Kit (Vazyme). The extraction steps were referred to the kit instructions. DNA enzyme, RNA enzyme, and water were added to the Xanthomonas citri phage XP1 nucleic acid extracted in the above steps. After reacting for 30 minutes, 5 μL was taken for agarose gel electrophoresis (see Figure 3 ).

[0057] The results showed that the phage nucleic acid could be degraded by DNase, indicating that the phage is a DNA phage. After verification of the DNA by gel electrophoresis, its concentration and purity were determined using a NanoDrop ND-1000. The product was sent to Guangdong Meige Gene Technology Co., Ltd. for sequencing. Xanthomonas citri subsp. citri phage XP1 has the nucleotide sequence shown in SEQ ID NO. 1.

[0058] 5. Biological Deposit

[0059] Xanthomonas phage XP1, deposit number: CCTCCNO: M 20241486, deposited by China Center for Type Culture Collection, Wuhan University, Wuhan, China; deposited on July 4, 2024.

[0060] Example 2 Detection of phage virulence gene or adverse gene deletion

[0061] The present invention selected 15 virulence genes identified as lysogenic phages from pathogenic bacteria (see Table 1), and determined whether the phage XP1 of Xanthomonas citri subsp. citri contained the above virulence genes by measuring the whole genome and performing bioinformatics analysis on it.

[0062] Table 1 Major known virulence genes of lysogenic phages in pathogenic bacteria

[0063]

[0064] The results showed that the tested phage of the present invention did not contain the above-mentioned virulence genes.

[0065] Example 3 Determination of the optimal multiplicity of infection (MOI) of Xanthomonas citri subsp. citri phage XP1.

[0066] A single colony of the host bacteria, Xanthomonas citri subsp. citri, was selected and inoculated into a conical flask containing 50 mL of NB culture medium. The culture was shaken at 200 rpm at 28°C for 48 hours to obtain a host bacterial suspension. Purified culture medium of Xanthomonas citri subsp. citri phage XP1 (prepared in Example 1) and the host bacteria were added at multiplicities of infection (MOI = number of phages / number of host bacteria) of 100, 10, 1, 0.1, 0.01, and 0.001, respectively. NB liquid medium was added to equalize the total volume in each tube. The culture was shaken at 200 rpm at 28°C for 12 hours. After completion of the culture, the tubes were centrifuged at 12,000 g for 10 minutes, and the supernatant was collected. The titer of each phage treated was determined using the double-layer plate method. The results are shown in Table 2. Three replicates were prepared for each dilution, and the average of the three replicates for that dilution was used for counting.

[0067] Each point was cultured in duplicate tubes and the average value was taken. The MOI that produced the highest phage titer was defined as the optimal multiplicity of infection. The experiment was repeated 3 times.

[0068] Table 2 Titers of Xanthomonas citri subsp. citri phage XP1 at different multiplicity of infection

[0069]

[0070] The results showed that when the MOI of Xanthomonas citri subsp. citri phage XP1 was 0.01, its titer reached the highest (1.13×10 9 PFU / mL). The optimal MOI of Xanthomonas citri subsp. citri phage XP1 was 0.01. This indicates that Xanthomonas citri subsp. citri phage XP1 can rapidly multiply into a large number of phages with only a very small amount of initial phage. This provides a high-quality source of phage strains for the industrial production of phage preparations for the prevention and treatment of citrus canker, and also provides a key production parameter for the multiplicity of infection of Xanthomonas citri subsp. citri phage XP1.

[0071] Example 4 Determination of the Thermal Stability of Xanthomonas citri subsp. citri Phage XP1

[0072] Take 1mL 1×10 9 Purified culture fluid of Xanthomonas citri subsp. citri phage XP1 (prepared in Example 1) was placed in sterile centrifuge tubes and incubated at -80°C, -20°C, 4°C, 28°C, 37°C, and 55°C for 3, 6, 12, and 24 hours, respectively. After incubation, the tubes were removed and immediately equilibrated at room temperature. After appropriate dilution, the phage potency was determined using the double-layer plate method. The experiment was repeated three times. The results are shown in Table 3.

[0073] Table 3 Stability of Xanthomonas citri subsp. citri phage XP1 at different temperatures

[0074]

[0075] As shown in Table 3, compared with the control, the titer of Xanthomonas citri subsp. citri phage XP1 was only reduced by one order of magnitude under the conditions of -80 to 28°C, indicating that Xanthomonas citri subsp. citri phage XP1 has good room temperature and low temperature storage properties.

[0076] Example 5 pH stability test of Xanthomonas citri subsp. citri phage XP1

[0077] 900 μL of NB medium with different pH values ​​(pH = 2-12) were added to sterile centrifuge tubes respectively. The centrifuge tubes were placed in a constant temperature water bath at 28°C. After the temperature was balanced, 100 μL of the purified culture medium of Xanthomonas citri subsp. citri phage XP1 (prepared in Example 1) was added to each of the sterile centrifuge tubes to make the initial titer 1×10 9 PFU / mL, incubated at 28°C for 2 h, and sampled for titer determination. The experiment was repeated three times. The results are shown in Table 4.

[0078] Table 4 Stability of Xanthomonas citri subsp. citri phage XP1 under different pH conditions

[0079] pH Phage titer (PFU / mL) 2 0 3 <![CDATA[2.00×10 5 ]]> 4 <![CDATA[4.67×10 6 ]]> 5 <![CDATA[7.33×10 8 ]]> 6 <![CDATA[7.33×10 8 ]]> 7 <![CDATA[4.80×10 8 ]]> 8 <![CDATA[3.80×10 8 ]]> 9 <![CDATA[5.33×10 8 ]]> 10 <![CDATA[5.73×10 8 ]]> 11 <![CDATA[1.07×10 8 ]]> 12 <![CDATA[1.33×10 7 ]]>

[0080] As shown in Table 4, the potency of Xanthomonas citri subsp. citri phage XP1 decreased by only one order of magnitude at most under the conditions of pH = 5-11, or even showed no significant change, indicating that the phage has good stability under neutral, acidic and alkaline conditions. Therefore, the present invention is hardly affected by the pH of the adjuvant when applied in the field.

[0081] Example 6 UV Stability Determination of Xanthomonas citri subsp. citri Phage XP1

[0082] Take 5mL 1×10 9 Purified culture fluid of Xanthomonas citri subsp. citri phage XP1 (prepared in Example 1) at 500 PFU / mL was plated onto a 90 mm diameter sterile Petri dish, placed in a clean bench, and irradiated under UV light. Samples were taken at 0, 5, 10, 15, 25, and 50 minutes, and placed in the dark for 30 minutes. Phage titer was then determined using the double-plate method. The results are shown in Table 5.

[0083] Table 5 UV stability determination of Xanthomonas citri subsp. citri phage XP1

[0084] Time (min) Phage titer (PFU / mL) 5 <![CDATA[1.00×10 9 ]]> 10 <![CDATA[1.07×10 9 ]]> 15 <![CDATA[1.13×10 9 ]]> 25 <![CDATA[1.07×10 9 ]]> 50 <![CDATA[1.00×10 9 ]]>

[0085] As shown in Table 5, after 50 minutes of ultraviolet irradiation, the potency of Xanthomonas citri subsp. citri phage XP1 did not change significantly. Therefore, the phage of the present invention has strong tolerance to ultraviolet rays and can be applied in the field without being affected by ultraviolet rays.

[0086] Example 7 Determination of the lysis spectrum of Xanthomonas citri subsp. citri phage XP1

[0087] The titer is 1×10 9 PFU / mL of the purified culture medium of Xanthomonas citri subsp. citri phage XP1 (prepared in Example 1) was used to determine the lysis spectrum of the test phage using a double-layer plate spot method. Single colonies of Xanthomonas citri subsp. citri were picked from 8 different counties in Hunan Province and inoculated into a conical flask containing 50mL of NB culture medium. The culture was shaken at 28°C and 200rpm for 48h to obtain bacterial cultures of each strain. 1mL of the test bacterial suspension was mixed with 5mL of NA semi-solid culture medium and spread on a NA plate. 10μL of XP1 phage was spotted on the plate. After air drying, the culture was cultured at 28°C for 12h, and the results were observed. The experiment was repeated 3 times. The results are shown in Table 6.

[0088] Table 6 Determination of the lysis spectrum of Xanthomonas citri subsp. citri phage XP1

[0089] strain number Cracking results CZ +++ YZ +++ KY + JH +++ HS + SM +++ MY +++ CL +

[0090] Note: “+++” means extremely strong cleavage; “++” means relatively strong cleavage; “+” means weak cleavage; “-” means no cleavage.

[0091] As shown in Table 6, Xanthomonas citri subsp. citri phage XP1 could effectively lyse 8 strains of Xanthomonas citri subsp. citri from different districts and counties, and had a wide lysis spectrum.

[0092] Example 8 Lysis Test of Xanthomonas citri subsp. citri Phage XP1 on Non-host Pathogenic Bacteria

[0093] Six single colonies of non-host pathogenic bacteria, including Xanthomonas campestris pv. campestris strains HF and CSG, Erwinia carotovora subsp. carotovora strains LXCC-NX and LXCC-TJ, Bacillus strain RF, and Dickeya dadantii 18020, were selected and inoculated into 50 mL of NB liquid medium in Erlenmeyer flasks. The cultures were shaken at 200 rpm at 28°C for 48 h to prepare bacterial suspensions. One mL of the test bacterial suspension was mixed with 5 mL of NA semisolid medium and plated on NA agar plates. 10 μL of Xanthomonas citri subsp. citri phage XP1 was spotted on the plates, air-dried and incubated at 28°C for 12 h. The results were observed. The experiment was repeated three times. The results are shown in Table 7.

[0094] Table 7 Lysis test of Xanthomonas citri subsp. citri phage XP1 against non-host pathogenic bacteria

[0095] Strain name Cracking results Xanthomonas campestris pv. campestris HF - Xanthomonas campestris pv. campestris CSG - Erwinia carotovora subsp. carrotus LXCC-NX - Erwinia carotovora subsp. carrotus LXCC-TJ - Bacillus RF - Dictyosporin 18020 -

[0096] Note: “-”: no lysis.

[0097] As shown in Table 7, Xanthomonas citri subsp. citri phage XP1 was unable to recognize any of the six non-host pathogenic bacteria tested, indicating that the tested phage has strong host specificity and has no damaging effect on other microbial communities.

[0098] Example 9: Control of Citrus Canker by Xanthomonas citri subsp. citri phage XP1

[0099] Select several new citrus leaves with the largest leaf tip area and green color as test materials. Wash the leaf surface with sterile water and select three areas on the front of the leaf for acupuncture treatment, with 3 needles in each area and 9 needles per leaf. Each treatment is repeated 3 times. Sterile filter paper is cut into discs. The size of the discs should be suitable for covering the three acupuncture points in the treated area of ​​the leaf. A single colony of Xanthomonas citri subsp. citri was inoculated into 50 mL of NB liquid medium and cultured with shaking at 200 rpm at 28°C for 48 hours. It was then diluted with sterile water to 1×10 8 CFU / mL of bacterial solution.

[0100] CK: Use sterile filter paper to absorb enough prepared Xanthomonas citri subsp. citri bacterial liquid, stick it on the three puncture points in the treated area of ​​each leaf, place the leaves in a culture dish moisturized with sterile filter paper, 3 leaves per dish, place the covered culture dish in a 28℃ incubator, open the culture dish cover regularly and spray sterile water to keep it moist. 7 days after inoculation, calculate the disease index of each citrus leaf.

[0101] Prevention group: Xanthomonas citri subsp. citri phage XP1 (prepared in Example 1) was diluted with sterile water to a concentration of 1×10 7 A solution of Xanthomonas citri subsp. citri phage XP1 at a concentration of 100 PFU / mL was evenly sprayed on the leaf surface. Two hours later, a sterile filter paper was used to absorb the prepared Xanthomonas citri subsp. citri phage solution and applied to three acupuncture points in the treated area of ​​each leaf. The leaves were then placed in a Petri dish moisturized with sterile filter paper, with three leaves per dish. The covered Petri dishes were placed in a 28°C incubator, and the lids were opened periodically to spray with sterile water for moisture. Seven days after inoculation, the disease index of each citrus leaf was calculated.

[0102] Treatment group: Use sterile filter paper to absorb the prepared Xanthomonas citri subsp. citri bacterial solution and apply it to the three acupuncture points in the treated area of ​​each leaf. After 2 hours, take Xanthomonas citri subsp. citri phage XP1 (prepared in Example 1) and dilute it with sterile water to a concentration of 1×10 7 A solution of Xanthomonas citri subsp. citri phage XP1 at a concentration of 100 PFU / mL was evenly sprayed on the leaf surface. The leaves were placed in sterile filter paper-lined Petri dishes, with three leaves per dish. The covered Petri dishes were placed in a 28°C incubator. The lids were opened periodically and sprayed with sterile water to keep them moist. Seven days after inoculation, the disease index of each citrus leaf was calculated.

[0103] The disease was graded according to the number of acupuncture points at which it developed: Grade 0, no acupuncture points at which it developed; Grade 1, 1 to 3 acupuncture points at which it developed; Grade 2, 4 to 6 acupuncture points at which it developed; and Grade 3, 7 to 9 acupuncture points at which it developed.

[0104] Disease index = ∑ (number of diseased leaves at each level × representative value of each level) / (total number of leaves surveyed × highest representative value) × 100.

[0105] Preventive effect = (CK disease index - treatment disease index) / CK disease index × 100%.

[0106] The disease index and control efficacy statistics for each treatment group are shown in Table 8. As shown in Table 8, the control efficacy of Xanthomonas citri subsp. citri phage XP1 against citrus canker in both the prevention and treatment groups reached 66.67%, indicating that Xanthomonas citri subsp. citri phage XP1 can be used as a biological agent to control citrus canker.

[0107] Table 8 The control effect of Xanthomonas citri subsp. citri phage XP1 on citrus canker

[0108] deal with Disease index Prevention effect CK 100 / Prevention Group 33.33 66.67％ Treatment group 33.33 66.67％

[0109] In summary, the Xanthomonas citri subspecies citrus bacteriophage XP1 of the present invention has good stability and high safety, can be used to prepare a kit, and can also be used as a biological disinfectant or biological pesticide, and can effectively prevent and treat bacterial diseases caused by Xanthomonas citri subspecies citrus.

[0110] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A Xanthomonas citri subsp. citri phage XP1, characterized in that: It was deposited in the China Center for Type Culture Collection on July 4, 2024, with the deposit address at Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M 20241486.

2. A use of the Xanthomonas citri subsp. citri phage XP1 according to claim 1 in the preparation of pesticides or fertilizers for preventing and treating citrus canker, characterized in that: The citrus canker is caused by Xanthomonas citri subsp. citri.

3. A pesticide or fertilizer for preventing and treating citrus canker, characterized in that: The active ingredient comprises the Xanthomonas citri subspecies citri phage XP1 according to claim 1.

4. The pesticide or fertilizer according to claim 3, characterized in that The pesticide or fertilizer further includes an adjuvant acceptable to the pesticide or fertilizer.

5. Use of the Xanthomonas citri subsp. citri phage XP1 according to claim 1 in the preparation of a disinfectant for Xanthomonas citri subsp. citri.

6. A disinfectant of Xanthomonas citri subsp. citri, characterized in that The active ingredient comprises the Xanthomonas citri subspecies citri phage XP1 according to claim 1.

7. The disinfectant according to claim 6, characterized in that The disinfectant is a liquid preparation.

8. The disinfectant according to claim 7, characterized in that The disinfectant includes auxiliary materials; the auxiliary materials include aqueous carriers.

9. The disinfectant according to claim 8, characterized in that The aqueous carrier is SM buffer, phosphate buffer, NB medium or LB medium.

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