Xanthomonas citri subsp.citri phage and application thereof

Abstract

The invention discloses xanthomonas citri subsp.citri phage and application thereof, and relates to the technical field of biology. The xanthomonas citrus subspecies phage is preserved in the China Center for Type Culture Collection on July 4, 2024, the preservation address is Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: M 20241486. The xanthomonas citrus subsp.citri bacteriophage can specifically inactivate the xanthomonas citrus subsp.citri, mass proliferation can be completed only with a small amount of initial bacteriophage, and a high-quality bacteriophage strain source is provided for industrial production of bacteriophage bactericides; technicists in the field can prepare various products applied to the aspects of detection, disinfection, plant protection and the like from the xanthomonas citrus subsp.citri phage provided by the invention according to records and common knowledge in the field by technicians in the field, and the products are industrially applied to the xanthomonas citrus subsp.citri phage.

Description

Technical Field

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

[0002] Xanthomonas citri subsp. citri is a gram-negative bacterium of the genus Xanthomonas. The bacteria are short rod-shaped, with blunt ends, 0.5-0.7×1.5-2.0μm in size, arranged singly, with a single polar flagellum, no spores, capsule, oxidase negative, and catalase positive. Xanthomonas citri subsp. citri appears as smooth yellow round colonies on NA medium, with neat, sticky, and slightly raised edges. The suitable growth temperature of the bacteria is 20-30℃, with a minimum of 5-10℃ and a maximum of 35℃. The lethal temperature is 55℃ for 10 minutes, and the optimum 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, mainly harming leaves, fruits and branches, forming corky, crater-like cracked lesions with raised sides. In the later stage, it can cause leaf fall, fruit drop and branch dieback, seriously reducing the yield and quality of citrus. Once infected, citrus trees carry the bacteria for life. The area of ​​citrus canker in my country exceeds 10 million mu each year, causing direct economic losses of more than 500 million yuan. At present, the prevention and control of citrus canker mainly relies on chemical pesticide copper preparations and antibiotics. Improper use of copper preparations can easily cause pesticide damage and lead to outbreaks of red spider mites. Long-term use of antibiotics can easily cause bacteria to develop drug resistance. Moreover, chemical pesticides are not selective. While killing pathogens, they also kill microorganisms that are beneficial to crops. Long-term and large-scale application of chemical agents also causes the accumulation of pesticide residues in plants, affecting 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 bacteriophages can complete adsorption, invasion, replication, assembly and cell lysis in a short period of time to kill bacteria. Since Frederik Tword first discovered bacteriophages in 1915, more and more studies have shown that bacteriophages have high antibacterial activity and specificity, and are not infectious to humans, other mammals, plants and non-target microorganisms. However, current research on bacteriophages is mostly focused on screening and the discussion of their biological characteristics, and there are few reports on their applications. Therefore, screening and applying suitable virulent bacteriophages is an effective way to develop new antibacterial agents.

[0005] At present, there is almost no research on Xanthomonas citri subsp. citrus phages, and there are few reports on the use of phage therapy to control citrus canker caused by Xanthomonas citri subsp. citrus. Therefore, it is of positive significance to develop Xanthomonas citri subsp. citrus phages for the prevention and treatment of related pathogens. Summary of the invention

[0006] The purpose of the present invention is to provide a Xanthomonas citri subspecies citrus bacteriophage and its application to solve the problems existing in the above-mentioned prior art. The Xanthomonas citri subspecies citrus bacteriophage can specifically inactivate Xanthomonas citri subspecies citrus, and only a small amount of initial phage is needed to complete a large number of proliferations, providing a high-quality phage strain source 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 bacteriophage (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 the use of the above-mentioned Xanthomonas citri subspecies citrus bacteriophage XP1 in the preparation of pesticides or fertilizers for preventing and treating citrus canker, wherein the citrus canker is caused by Xanthomonas citri subspecies citrus.

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

[0011] Furthermore, the pesticide or fertilizer also includes auxiliary materials acceptable to the pesticide or fertilizer.

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

[0013] The present invention also provides a disinfectant of Xanthomonas citri subspecies citrus, wherein the active ingredient comprises the above-mentioned Xanthomonas citri subspecies citrus bacteriophage 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 the use of the Xanthomonas citri subspecies citrus bacteriophage XP1 in preparing a detection product of Xanthomonas citri subspecies citrus.

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

[0019] (1) The bacteriophage XP1 of Xanthomonas citri subsp. citri 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 lysis ability. The optimal MOI can reach 1.13×10 within 12 h. 9 PFU / mL or above. Xanthomonas citri subsp. citri phage XP1 can specifically inactivate Xanthomonas citri subsp. citri phage, and only a small amount of initial phage is needed to complete a large number of proliferations, providing a high-quality phage strain source for the industrial production of phage fungicides; those skilled in the art can prepare the Xanthomonas citri subsp. citri phage XP1 provided by the present invention into various products for detection, disinfection, plant protection, etc. and apply them industrially according to the records of the present invention and the common sense in the field.

[0021] (3) The Xanthomonas citri subsp. citri phage XP1 of the present invention is a strictly virulent phage and has high specificity and lysis for host bacteria and a wide host range. The lysis rate of typical Xanthomonas citri subsp. citri from 8 different counties is as high as 100%. Xanthomonas citri subsp. citri phage XP1 can be used as an effective ingredient in various products for environmental disinfection, such as, but not limited to, disinfection of water distribution systems, irrigation facilities, public and private facilities or other environmental surfaces in the form of liquid immersion, spraying, and combined use with aqueous carriers, which can effectively control the growth and activity of target bacteria. Liquid immersion and spraying 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) The bacteriophage XP1 of Xanthomonas citri subsp. citri has good stability. Under the condition of -80~28℃, the potency can be reduced by no more than one order of magnitude within 24 hours. Under the condition of pH=5~11, the stability is good, and the potency 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, which 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 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 the common knowledge in the art into a biological agent that can be used to prevent and treat 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 drawings required for use in the embodiments will be briefly introduced below. 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 creative work.

[0027] Figure 1 The phage plaque image is 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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

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

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 a conflict with any incorporated document, the content 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 of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary 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 storage of Xanthomonas citri subsp. citri bacteriophage XP1

[0040] The source sample of Xanthomonas citri subsp. citri phage XP1 in the present invention is collected from the peel of the orange orchard in Yizhang County, Chenzhou City, Hunan Province. The peel is homogenized with water and filtered through double-layer filter paper. The filtrate is centrifuged at low speed and room temperature, and then the supernatant is 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 logarithmic phase host bacterial culture, and culture at 28°C with shaking at 200 rpm 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 for later use.

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

[0045] 2. Purification of phage

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

[0047] (2) Add 5 mL of semisolid NA medium at no higher than 45°C to 0.5 mL of logarithmic-phase host bacterial solution, mix well 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 h and observe it. A double-layer plate containing a single plaque is obtained.

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

[0050] (5) A single plaque with the same size and shape was 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 min, and the supernatant was filtered through a 0.22 μm filter membrane to obtain a purified phage solution, namely, Xanthomonas citri subsp. citri phage XP1. Xanthomonas citri subsp. citri phage XP1 produced a single circular plaque on the bacterial lawn of Xanthomonas citri subsp. citri phage XP1, which was clear in the center and 4-5 mm in diameter (see Figure 1 ).

[0052] 3. Electron microscopic observation of bacteriophage

[0053] Take the purified Xanthomonas citri subsp. citri phage XP1 solution for electron microscopy observation: take 20 μL of the sample and drop it on the copper grid, wait for it to precipitate naturally for 15 minutes, absorb the excess liquid from the side with filter paper, add 1 drop of 2% phosphotungstic acid (PTA) on the copper grid, stain for 10 minutes, absorb the dye from the side with filter paper, and observe it under transmission electron microscopy after drying.

[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 inextractable 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] Take 200 μL of the purified Xanthomonas citri phage XP1 solution and use the FastPureViral DNA / RNA Mini Kit Viral DNA / RNA Extraction Kit (Vazyme) to extract the phage nucleic acid. The extraction steps refer to the instructions of the kit. Take the Xanthomonas citri phage XP1 nucleic acid extracted in the above steps and add DNase, RNase and water respectively. After reacting for 30 minutes, take 5 μL 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 was a DNA phage. After gel electrophoresis to verify the DNA, the concentration and purity were determined using 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, the deposit number is CCTCCNO: M 20241486, the depository is China Center for Type Culture Collection, address is Wuhan University, Wuhan, China; the deposit time is July 4, 2024.

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

[0061] The present invention selects 15 virulence genes identified as being derived from lysogenic phages in pathogenic bacteria (see Table 1), and determines whether the phage XP1 of Xanthomonas citri subsp. citri contains 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 gene.

[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 picked up and inoculated into a conical flask containing 50 mL of NB culture solution. The culture was shaken at 200 rpm for 48 h at 28°C to obtain a host bacteria suspension. The purified culture solution of Xanthomonas citri subsp. citri phage XP1 (prepared in Example 1) and the host bacteria (MOI = number of phages / number of host bacteria) were added at a multiplicity of infection of 100, 10, 1, 0.1, 0.01 and 0.001, respectively. NB liquid culture medium was added to make the total volume of each tube the same, and the culture was shaken at 200 rpm for 12 h at 28°C. After the culture was completed, the supernatant was collected by centrifugation at 12000g for 10 min, and the titer of each treated phage was determined by the double-layer plate method. The results are shown in Table 2. Three parallel samples were required for each dilution, and the average of the three parallel samples of this dilution was taken for counting.

[0067] Each point was cultured in duplicate tubes and the average value was taken, and the MOI that produced the highest phage titer was taken 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 infection multiplicities

[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 is 0.01. This indicates that Xanthomonas citri subsp. citri phage XP1 can rapidly proliferate into a large number of phages with only a very small amount of initial phages, providing a high-quality phage strain source for the industrial production of phage preparations for the prevention and treatment of citrus canker, as well as the infection multiplicity of Xanthomonas citri subsp. citri phage XP1, an important production parameter.

[0071] Example 4 Determination of the thermal stability of Xanthomonas citri subsp. citri phage XP1

[0072] Take 1 mL 1×10 9 PFU / mL Xanthomonas citri subsp. citri phage XP1 purified culture fluid (prepared in Example 1) was placed in a sterile centrifuge tube and placed under -80°C, -20°C, 4°C, 28°C, 37°C, and 55°C for 3h, 6h, 12h, and 24h, respectively. After the action time, the centrifuge tube was taken out and immediately placed at room temperature for equilibrium. After appropriate dilution, the phage efficacy was determined by the double-layer plate method. The experiment was repeated 3 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 at -80 to 28°C was only reduced by 1 order of magnitude, indicating that Xanthomonas citri subsp. citri phage XP1 has good room temperature and low temperature storage characteristics.

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

[0077] Take sterile centrifuge tubes and add 900 μL of NB medium with different pH (pH = 2-12) respectively. Place the above centrifuge tubes in a constant temperature water bath at 28°C. After the temperature is balanced, add 100 μL of the purified culture solution of Xanthomonas citri subsp. citri phage XP1 (prepared in Example 1) to make the initial titer of 1×10 9 PFU / mL, let it stand at 28℃, take samples and measure the titer after 2h of reaction, repeat the experiment 3 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 at pH = 5-11 only decreases by one order of magnitude at most or even has no obvious change, indicating that the phage has good stability under neutral, acidic and alkaline conditions. Therefore, the present invention, if applied in the field, is hardly affected by the pH of the adjuvant.

[0081] Example 6 UV stability determination of Xanthomonas citri subsp. citri phage XP1

[0082] Take 5mL 1×10 9 PFU / mL Xanthomonas citri subsp. citri phage XP1 purified culture fluid (prepared in Example 1) was spread in a 90 mm diameter sterile culture dish, placed in a clean bench, and irradiated under ultraviolet light. Samples were taken at 0 min, 5 min, 10 min, 15 min, 25 min, and 50 min, respectively, and placed in a dark place for 30 min. After that, the phage titer was determined by the double-layer plate method. The results are shown in Table 5.

[0083] Table 5 UV stability test 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 a 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 Xanthomonas citri subsp. citri phage XP1 purified culture fluid (prepared in Example 1), the double-layer plate spot method was used to determine the lysis spectrum of the test phage. Single colonies of Xanthomonas citri subsp. citri from 8 different counties in Hunan Province were picked respectively, inoculated into a conical flask containing 50mL NB culture medium, and cultured at 28℃ and 200rpm for 48h to obtain bacterial culture fluids of various strains. Take 1mL of the test bacterial suspension and mix it with 5mL NA semi-solid culture medium and spread it on a NA plate, take 10μL XP1 phage and spot it on the plate. After air drying, culture at 28℃ for 12h, observe the results, and repeat the experiment 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 single cleavage.

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

[0092] Example 8 Lysis test of Xanthomonas citri subsp. citri phage XP1 on non-host pathogenic bacteria

[0093] Six strains of non-host pathogenic bacteria were selected, including Xanthomonas campestris pv. campestris strain HF, Xanthomonas campestris pv. campestris strain CSG, Erwinia carotovora subsp. carotovora strain LXCC-NX, Erwinia carotovora subsp. carotovora strain LXCC-TJ, Bacillus strain RF, and Dickeya dadantii 18020, and inoculated into conical flasks containing 50 mL NB liquid culture medium, and cultured at 28°C and 200 rpm for 48 hours to obtain bacterial suspensions of each strain. 1 mL of the test bacterial suspension was mixed with 5 mL of NA semi-solid culture medium and spread on NA agar plates. 10 μL of the bacteriophage liquid of Xanthomonas citri subsp. citri was spotted on the plate, and then air-dried and cultured at 28°C for 12 h. The results were observed. The experiment was repeated 3 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. carrot LXCC-TJ - Bacillus RF - Dictyosporin 18020 -

[0096] Note: “-”: no lysis.

[0097] As shown in Table 7, the Xanthomonas citri subsp. citri phage XP1 could not 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 Phage XP1 of Xanthomonas citri subsp. citri

[0099] Select several new citrus leaves with the largest tip area and green color as test materials, wash the leaf surface with sterile water, select three areas on the front of the leaf for acupuncture treatment, 3 needles in each area, 9 needles per leaf, and set 3 replicates for each treatment. Cut the sterile filter paper into discs, the size of which should cover the 3 acupuncture points in the leaf treatment area. Inoculate a single colony of Xanthomonas citri subsp. citri into 50 mL NB liquid culture medium, shake and culture at 200 rpm for 48 h at 28 ° C, and prepare it into 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 pricking points in the treated area of ​​each leaf, place the leaves in a culture dish moisturized by sterile filter paper, 3 leaves in each dish, place the covered culture dish in a 28℃ incubator, open the culture dish cover regularly and spray sterile water to keep it moist, and calculate the disease index of each citrus leaf 7 days after inoculation.

[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 PFU / mL of Xanthomonas citri subsp. citri phage XP1 solution, spray the phage solution evenly on the leaf surface. After 2 hours, use a sterile filter paper to absorb the prepared Xanthomonas citri subsp. citri subsp. citri phage solution, stick it on the three acupuncture points in the treatment area of ​​each leaf, place the leaves in a sterile filter paper moisturizing culture dish, 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, and count the disease index of each citrus leaf 7 days after inoculation.

[0102] Treatment group: Use sterile filter paper to absorb the prepared Xanthomonas citri subsp. citri bacterial liquid and stick it on the three acupuncture points in the treatment 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 PFU / mL of Xanthomonas citri subsp. citri phage XP1 solution, spray the phage solution evenly on the leaf surface. Place the leaves in a sterile filter paper moisturizing petri dish, 3 leaves per dish, place the covered petri dish in a 28℃ incubator, open the petri dish cover regularly and spray sterile water to keep it moist, and count the disease index of each citrus leaf 7 days after inoculation.

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

[0104] Disease index = ∑ (number of diseased leaves at each level × representative value at 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 of each treatment group are shown in Table 8. As shown in Table 8, the control efficacy of Xanthomonas citri subsp. citrus phage XP1 against citrus canker in both the prevention group and the treatment group can reach 66.67%, indicating that Xanthomonas citri subsp. citrus phage XP1 can be used as a biological agent to prevent and control citrus canker.

[0107] Table 8 The protective 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 only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined 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 as claimed in 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 described in claim 1.

4. The pesticide or fertilizer according to claim 3, characterized in that: The pesticide or fertilizer also includes pesticide or fertilizer acceptable adjuvants.

5. Use of the Xanthomonas citri subsp. citri phage XP1 as claimed in 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 described in 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 comprises auxiliary materials; the auxiliary materials comprise an aqueous carrier.

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

10. Use of the Xanthomonas citri subsp. citri phage XP1 as claimed in claim 1 in preparing a detection product for Xanthomonas citri subsp. citri.

Images