Phage of pseudomonas syringae pv actinidiae and use thereof

Abstract

The present application relates to the technical field of bacteriophage, and discloses a bacteriophage of Pseudomonas syringae pv. actidii and application thereof, and the preservation number of the bacteriophage is CGMCC No. 45227. The bacteriophage provided by the present application can specifically lyse Pseudomonas syringae pv. actidii, and therefore, the bacteriophage can be used for preventing and treating bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial bacterial

Description

Technical Field

[0001] This invention relates to the field of bacteriophage technology, specifically to a bacteriophage of *Pseudomonas syringae* pathogenic species in kiwifruit and its application. Background Technology

[0002] *Pseudomonas syringae* pv. *actinidiae* (Psa) is the pathogen of bacterial canker in kiwifruit. Symptoms of canker include brown leaf spots, leaf blight, stem tip death, pus discharge from the trunk, and fruit rot, which can lead to widespread plant death in severe cases. The disease was first reported in China and Japan in the 1980s, and experienced a severe outbreak in Italy in 2008, spreading to other European countries and seriously threatening the global kiwifruit industry. In recent years, the losses caused by canker to my country's kiwifruit industry have become increasingly serious, becoming a critical problem that urgently needs to be solved.

[0003] Currently, the most common method for preventing and controlling bacterial canker in kiwifruit is to spray the orchard with disinfectants, copper-based fungicides, or antibiotics. These chemical agents have problems such as high residues in the environment and food, and bacterial resistance. Therefore, it is of great significance to develop safe and efficient methods to prevent and control bacterial canker in kiwifruit. Summary of the Invention

[0004] The purpose of this invention is to overcome the problems of current methods for preventing and controlling bacterial canker in kiwifruit, which mainly rely on chemical agents and have high residues and bacterial resistance in the environment and food. This invention provides a bacteriophage of *Pseudomonas syringae* pathogenic species in kiwifruit and its application.

[0005] To achieve the above objectives, the present invention provides a bacteriophage of *Pseudomonas syringae* pathogenic species in kiwifruit, the bacteriophage having the accession number CGMCC No. 45227.

[0006] Preferably, the bacteriophage is resistant to environments with a pH value of 4 to 11.

[0007] Preferably, the bacteriophage is resistant to temperatures of 28–50°C.

[0008] Preferably, the optimal multiplicity of infection for the bacteriophage is 0.01.

[0009] A second aspect of the present invention provides the use of the bacteriophage described above in the preparation of a biological agent for the prevention and treatment of bacterial canker in kiwifruit.

[0010] Preferably, the dosage form of the biological agent is a spray or an injection.

[0011] Preferably, the injection site is the phloem of the kiwifruit tree to be treated.

[0012] Preferably, the preparation process of the biopharmaceutical includes: the preparation process of the biopharmaceutical includes: preparing a solution with a titer of 10... 8 ~10 9 PFU / mL phage suspension was mixed with water and Tween.

[0013] Preferably, the volume ratio of the phage suspension to water is 1:20 to 40.

[0014] Preferably, the volume fraction of Tween in the biological agent is 0.02-0.1%.

[0015] The bacteriophage provided by this invention can specifically lyse *Pseudomonas syringae* pv. *actinidiae* (Psa). Therefore, this bacteriophage can be used to prevent and control bacterial canker in kiwifruit caused by *Pseudomonas syringae* psa. At the same time, this invention can expand the bacteriophage resource library of *Pseudomonas syringae* psa, which is of great significance in the strategic layout for the prevention and control of kiwifruit canker. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of the morphology of phage plaques on a double-layer plate provided by the present invention;

[0017] Figure 2 This is a schematic diagram of the transmission electron microscopy results of the bacteriophage provided by the present invention;

[0018] Figure 3 This is a graph showing the test results of the bacteriophage provided by this invention at different temperatures;

[0019] Figure 4 This is a graph showing the test results of the bacteriophage provided by this invention at different pH levels;

[0020] Figure 5 This is a schematic diagram of the one-step growth curve of the bacteriophage provided by the present invention.

[0021] Biological Preservation

[0022] The bacteriophage provided by this invention is a virulent bacteriophage isolated from nature. This bacteriophage was deposited on July 25, 2022, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 45227 and classification name: Pseudomonas syringae bacteriophage KBC54. Detailed Implementation

[0023] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0024] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0025] This invention provides a bacteriophage of *Pseudomonas syringae* pathogenic species in kiwifruit, the preservation number of which is CGMCC No. 45227.

[0026] The bacteriophage provided by this invention, as a virus that specifically infects pathogenic strains of *Pseudomonas syringae* in kiwifruit, generally does not affect the normal microbiota and can safely and effectively combat drug resistance of pathogenic strains of *Pseudomonas syringae* in kiwifruit.

[0027] Furthermore, during the interaction between bacteria and bacteriophages, due to the genetic diversity of *Pseudomonas syringae* pathogenic strains in kiwifruit, and the fact that different bacteriophages specifically recognize different strains, the characteristics of their co-evolution are related to the interaction. Therefore, to ensure the diversity and coverage of bacteriophages, a rich bacteriophage resource library targeting *Pseudomonas syringae* pathogenic strains in kiwifruit is needed. The bacteriophages provided by this invention can expand the bacteriophage resource library for *Pseudomonas syringae* pathogenic strains in kiwifruit, and are of great significance in the strategic layout for the prevention and control of kiwifruit canker disease.

[0028] The bacteriophage described in this invention has good stability. Specifically, the bacteriophage is resistant to pH values ​​of 4 to 11, and its titer decreases by no more than three orders of magnitude within 30 minutes.

[0029] In this invention, the bacteriophage is resistant to temperatures of 28–50°C, and its titer decreases by no more than two orders of magnitude within 30 minutes.

[0030] In a specific implementation, the optimal multiplicity of infection for the bacteriophage is 0.01.

[0031] This invention also proposes the application of the bacteriophage preparation described above in the preparation of biological agents for the prevention and treatment of bacterial canker in kiwifruit.

[0032] This invention does not limit the formulation of the biological agent, as long as it can come into contact with *Pseudomonas syringae* pathogenic species in kiwifruit trees suffering from bacterial canker. In specific embodiments, the biological agent is in the form of a spray or an injection. Preferably, both spraying and injection are used simultaneously to control the disease in kiwifruit trees.

[0033] In order to ensure that the biological agent has a good control effect on kiwifruit trees, in a preferred embodiment, the injection site of the injection is the phloem of the kiwifruit tree to be controlled.

[0034] In a specific embodiment, the preparation process of the biopharmaceutical includes: preparing a reagent with a titer of 10... 8 ~10 9 PFU / mL phage suspension was mixed with water and Tween.

[0035] In a preferred embodiment, the volume ratio of the phage suspension to water is 1:20 to 40, specifically, for example, 1:20, 1:22, 1:25, 1:30, 1:35, 1:39 or 1:40.

[0036] In a preferred embodiment, the volume fraction of Tween in the biological agent is 0.02-0.1%.

[0037] In the most preferred embodiment, a titer of 10 is prepared at a volume ratio of 1:39. 9 The PFU / mL phage suspension was mixed with the mixture, and the volume fraction of Tween in the biopharmaceutical was 0.05%.

[0038] The present invention will be described in detail below through embodiments, but the scope of protection of the present invention is not limited thereto. In the following embodiments, the host bacterium is *Pseudomonas syringaepv. actinidiae* (Psa), which was isolated from diseased plants in a kiwi orchard in Cangxi County, Sichuan Province.

[0039] Example 1

[0040] This embodiment is used to illustrate the isolation and purification of the bacteriophage of the present invention.

[0041] The wastewater samples used in this invention were collected from the Institute of Microbiology, Chinese Academy of Sciences;

[0042] The collected wastewater samples were centrifuged at 7,500×g for 5 minutes, and the supernatant was collected and then filtered through a 0.22μm filter membrane for sterilization to obtain a water sample.

[0043] (1) Isolation of bacteriophages

[0044] The host bacteria were cultured to the logarithmic growth phase to obtain a bacterial suspension. 50 μL of the bacterial suspension was added to 1 mL of water sample, mixed thoroughly, and allowed to stand at room temperature for 15 min. Then, it was added to 3 mL of LB medium (upper layer) at 42 °C and mixed thoroughly to obtain a mixed suspension. The mixed suspension was poured into LB medium (lower layer) containing 1.5% agar (double-layer plate method). After solidification, it was incubated at 28 °C overnight, and the phage plaques on the plates were observed after overnight incubation.

[0045] (2) Purification of bacteriophages

[0046] Transfer the newly grown single phage plaques to sterile 1.5 mL centrifuge tubes, crush them with a toothpick, add 1 mL of ddH2O and mix well. Incubate overnight at 4°C, then filter through a 0.22 μm filter membrane for sterilization and perform serial dilutions. Take the stock solution, 10... -1 10 -2 200 μL of each diluted phage was mixed with 50 μL of host bacterial culture and cultured using the double-layer plate method to obtain double-layer plates containing a single phage plaque.

[0047] After repeating the purification process 3-4 times, purified phages were obtained (see...). Figure 1 It was named bacteriophage KBC54.

[0048] Example 2

[0049] This embodiment is used to illustrate the identification of the bacteriophage of the present invention.

[0050] (1) Morphological observation of bacteriophages

[0051] The bacteriophage KBC54 obtained in Example 1 was observed under a transmission electron microscope, and the results are as follows: Figure 2 As shown. By Figure 2 It can be seen that the head of this bacteriophage is polyhedral symmetrical, with a diameter of about 58 nm, and the tail is about 12 nm. According to the classification standard of bacteriophages, bacteriophage KBC54 belongs to the family Brachyphageidae of the order Tailed Phages.

[0052] (2) Phage sequencing

[0053] The stock solution of phage KBC54 obtained in Example 1 was sent to Shanghai Ling'en Biotechnology Co., Ltd. for sequencing analysis using Illumina TruSeq. TMLibrary construction was performed using the Nano DNA Sample Prep Kit method. GeneMarks software was used to predict coding genes in the newly sequenced genome. The protein sequences of the predicted genes were compared with databases such as NR, Swiss-Prot, eggNOG, KEGG, and GO to obtain annotation information. Sequencing results showed that the isolated phage genome was a circular double-stranded DNA, 42.609 kb in size, with a GC content of 63.1%. Gene annotation results showed a total of 47 open reading frames (ORFs), of which 17 genes encoded hypothetical proteins, 7 encoded endonucleases, RNases, and DNases related to the regulatory function of phage genetic material, and the remainder were genes related to phage structure and function, encoding head proteins, capsid proteins, tail tube proteins, and other proteins. No tRNAs were predicted using tRNAscan-SE.

[0054] The phage genome sequences obtained from sequencing were compared with the NCBI database. Based on BlastN search results and phage classification principles (the threshold for phage genus-level classification is at least 50% nucleotide sequence similarity, and the threshold for species-level classification is at least 95% nucleotide sequence identity), we identified the isolated phage as a new species belonging to the class Caudoviricetes, order Caudovirales, family Autographiviridae, and genus Pollyceevirus. The genome sequence of Psa phage KBC54 has been submitted to GenBank (accession number: OL854071), and the complete genome sequence is shown in SEQ ID NO: 1.

[0055] Example 3

[0056] This embodiment illustrates the effect of temperature on the phage titer of the present invention.

[0057] With a titer of approximately 10 8 A 500 μL suspension of phage KBC54 (obtained in Example 1) at PFU / mL was incubated in water baths at 28°C, 50°C, 60°C, 70°C, and 80°C for 30 min, followed by rapid cooling on ice. The cooled phage KBC54 suspension was diluted 10-fold and mixed with host bacteria. The phage KBC54 was counted using the bilayer agar method. Based on the counting results, a curve showing the effect of temperature on the phage KBC54 titer was plotted with temperature on the x-axis and the logarithm of the phage titer on the y-axis. (See figure). Figure 3 .

[0058] Depend on Figure 3It can be seen that after bacteriophage KBC54 was treated at 28–50℃ for 30 min, the titer did not change significantly, and the activity remained high; after treatment at 60–70℃, the titer decreased by 10%. 5 When the temperature exceeds 80°C, bacteriophage KBC54 is completely inactivated. This demonstrates that the bacteriophage KBC54 provided by this invention is resistant to temperatures between 28 and 50°C.

[0059] Example 4

[0060] This example illustrates the effect of pH on the phage titer of the present invention.

[0061] In a 1.5 mL EP tube, first add 900 μL of LB liquid culture medium at different pH values ​​(2, 4, 6, 7, 8, 10, 12), then add 100 μL of a titer of approximately 10. 9 A PFU / mL suspension of phage KBC54 (obtained in Example 1) was placed in a 28°C water bath for 30 min. The phage KBC54 suspension was diluted 10-fold and mixed with host bacteria. Phage counts were performed using the double-layer plate method. Based on the count results, a curve showing the effect of pH on the titer of phage KBC54 was plotted with pH as the x-axis and the logarithm of the phage titer as the y-axis. (See figure). Figure 4 .

[0062] Depend on Figure 4 It can be seen that when the pH is 6–10, the phage concentration can be maintained at 10. 8 Approximately PFU / mL; when pH was 4 or 11, the phage titer decreased by 10. 2 However, at pH 2 or 12, the bacteriophage is completely inactivated. This demonstrates that the bacteriophage KBC54 provided by this invention is resistant to strong acids and alkalis.

[0063] Example 5

[0064] This embodiment illustrates the optimal multiplicity of infection of the bacteriophage of the present invention against the host bacterium (Pseudomonas syringae, a pathogenic species of Actinopterygium kiwifruit).

[0065] Phage KBC54 and host bacteria were added to 100 mL of KB liquid medium with multiplicity of infection of 1, 0.1, 0.01 and 0.001, and cultured at 28 °C and 220 rpm for 24 h. The supernatant after centrifugation was sterilized with a 0.22 μm filter membrane, and the titer of phage was determined by the double-layer plate method. The results are shown in Table 1 below.

[0066] Table 1

[0067] Host bacteria (CFU / mL) Bacteriophage (PFU / mL) Multiple infection Phage titer (PFU / mL) <![CDATA[1×10 4 ]]> <![CDATA[1×10 4 ]]> 1 <![CDATA[7.2×10 6 ]]> <![CDATA[1×10 5 ]]> <![CDATA[1×10 4 ]]> 0.1 <![CDATA[3.7×10 7 <!-- 4 -->]]> <![CDATA[1×10 6 ]]> <![CDATA[1×10 4 ]]> 0.01 <![CDATA[4.2×10 8 ]]> <![CDATA[1×10 7 ]]> <![CDATA[1×10 4 ]]> 0.001 <![CDATA[2.4×10 8 ]]>

[0068] As shown in Table 1, the phage titer increases as the multiplicity of infection (MDI) decreases from 1 to 0.1. The phage titer is highest at an MDI of 0.01, and then decreases as the MDI decreases to 0.001. This indicates that 0.01 is the optimal MDI for phage KBC54.

[0069] Example 6

[0070] This embodiment is used to illustrate the one-step growth curve of the bacteriophage of the present invention.

[0071] The phage KBC54 obtained in Example 1 was mixed with the host bacteria at a multiplicity of infection (MOI) of 0.01 and incubated at room temperature for 20 min. Then, it was centrifuged at 13,000 × g for 1 min, the supernatant was discarded, and the sample was washed twice with 1 mL of LB medium. The pellet was then resuspended in 5 mL of LB medium and incubated at 28 °C and 220 rpm. Starting from 0 min, 300 μL samples were taken every 10 min, centrifuged at 13,000 × g for 1 min, and then 100 μL of the supernatant was serially diluted 10-fold. The titer of phage KBC54 was determined using the double-layer plate method. Based on the results, a one-step growth curve was plotted with time on the x-axis and the titer of phage KBC54 on the y-axis. (See figure). Figure 5 .

[0072] Depend on Figure 5 It can be seen that the titer of phage KBC54 did not increase significantly in the first 20 minutes, which is the incubation period of the phage. From 20 to 70 minutes, the titer of phage KBC54 increased rapidly, which is the lysis period of the phage. After 70 minutes, the titer of phage tended to stabilize, which indicates that the phage entered the plateau phase. The burst size of this phage was 702.

[0073] Example 7

[0074] This embodiment illustrates the preventive and therapeutic effects of the bacteriophage of the present invention on bacterial canker in kiwifruit.

[0075] An experiment was conducted in four kiwifruit orchards in Cangxi County, Sichuan Province, where kiwifruit cultivars were severely affected. The kiwifruit variety used was "Hongyang" red-fleshed kiwifruit. The orchards were divided into a control group and a treatment group, with 50 plants in each group. A titer of 10... 9 A 1L suspension of phage KBC54 (obtained in Example 1) at PFU / mL was added to 39L of water, followed by the addition of Tween to obtain a biological agent (containing 0.05% Tween). This biological agent was used for spraying and phloem infusion on the entire kiwifruit plant for control. Application was performed in December of the current year, and in February and March of the following year, for a total of three applications. The control group received an equal volume of water containing 0.05% Tween. The incidence of canker was observed one month after the last treatment.

[0076] The results showed that, compared with the control group, the disease index (disease index = prevalence × severity × 100) of kiwifruit trees treated with phage KBC54 decreased by 50%, and the fruit set rate of kiwifruit increased by 30%, indicating that the phage KBC54 provided by the present invention has a good inhibitory effect on bacterial canker of kiwifruit.

[0077] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A bacteriophage that lyses a pathogenic strain of *Pseudomonas syringae* in kiwifruit, characterized in that, The phage has the accession number CGMCC No. 45227.

2. The bacteriophage according to claim 1, characterized in that, The bacteriophage is resistant to environments with a pH value of 4 to 11.

3. The bacteriophage according to claim 1, characterized in that, The bacteriophage is resistant to temperatures ranging from 28 to 50°C.

4. The bacteriophage according to claim 1, characterized in that, The optimal multiplicity of infection for the bacteriophage is 0.

01.

5. The use of a bacteriophage according to any one of claims 1-4 in the preparation of a biological agent for the prevention and treatment of bacterial canker in kiwifruit.

6. The application according to claim 5, characterized in that, The dosage form of the biological agent is a spray or an injection.

7. The application according to claim 6, characterized in that, The injection site is the phloem of the kiwifruit tree to be treated.

8. The application according to claim 5, characterized in that, The preparation process of the biopharmaceutical includes: [the following steps are described in the original text, which is not directly related to the preparation process] 8 ~10 9 PFU / mL phage suspension was mixed with water and Tween.

9. The application according to claim 8, characterized in that, The volume ratio of the phage suspension to water is 1:20~40.

10. The application according to claim 8, characterized in that, In the biological agent, the volume fraction of Tween is 0.02~0.1%.

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