Citrus canker phage SAC and application thereof
By developing citrus canker phage SAC, the negative impact of traditional chemical control methods on the environment and ecology has been solved, and efficient and environmentally friendly citrus canker prevention and treatment effects have been achieved.
Patent Information
- Application Number
- CN202510162591.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-14
- Publication Date
- 2025-06-10
AI Technical Summary
The prior art relies on copper-based compounds and antibiotics in the prevention and treatment of citrus canker diseases, which leads to environmental and ecological problems, and long-term use may cause drug resistance problems.
A citrus canker phage SAC was developed, which can effectively cleave the citrus subspecies of E. citrus, providing a new biological control method.
By using citrus canker phage SAC, the incidence of citrus canker disease can be effectively reduced, the yield loss caused by the disease canker disease canker disease can be reduced, and chemical pesticides can be avoided to protect the ecological environment and consumer health.
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Figure CN120118855A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of microbial applications, and particularly to a citrus canker phage SAC and its application. Background Art
[0002] Citrus canker is a serious disease caused by the pathogenic bacterium Xanthomonas citri subsp. citri (Xcci), which poses a major challenge and threat to the production of citrus fruits worldwide. Xcci invades citrus plants through stomata or wounds, and then colonizes and multiplies in the cytoplasmic interstices, resulting in the shedding and withering of citrus leaves, the gradual weakening of the tree body, and even a large amount of fruit shedding in severe cases, seriously affecting the yield and quality of citrus fruits and bringing huge economic losses to fruit farmers.
[0003] At present, the control of citrus canker mainly relies on the use of copper-based compounds and antibiotics. However, while these methods effectively control the disease, they also bring a series of environmental and ecological problems. Copper ions in copper-based compounds are toxic to plants themselves. Long-term use will not only affect the normal growth of plants, but may also have a negative impact on pollinating insects and other beneficial organisms, disrupting the ecological balance. In addition, the bioaccumulation of copper ions in soil and surface water will lead to a reduction in microbial biodiversity, further exacerbating environmental pressure. The use of antibiotics, although not accumulating in plants, will change the microbial community structure in the surrounding environment, stimulate the development of resistant traits in bacteria, and may cause more serious drug resistance problems in the long run.
[0004] In view of the limitations of traditional chemical control methods, it is particularly important to find more environmentally friendly and efficient disease control means. As a virus that can accurately identify and lyse specific bacteria, phage provides a new idea for biological control of diseases. Phages have a high degree of specificity and can targetedly lyse host bacteria without affecting other non-target organisms, so they have high biological safety in use. Compared with traditional chemical pesticides, phages have less impact on the environment and non-target organisms, which is more in line with the development concept of modern ecological agriculture.
[0005] In addition, phages also have the ability to self-replicate. They can continuously replicate using pathogenic bacteria as hosts. This characteristic enables phages to continuously play a bactericidal role during application, reducing the use cost and improving the control effect. At present, some substantial progress has been made in the application of phages in the field of agricultural disease control. For example, the American company Omnilytics successfully developed a phage pesticide product, which was successfully approved by the US Environmental Protection Agency and entered the market for sale, further proving the great potential and application value of phages as new pesticides.
[0006] Therefore, the present invention aims to provide a phage SAC against the pathogenic bacterium Xcci of citrus canker, and its application in improving the biological control effect of citrus canker, in order to provide a new, more environmentally friendly and efficient biological control means for solving the problem of controlling citrus canker. Summary of the Invention
[0007] In view of this, the present invention provides a citrus canker phage (Caudoviales phage) SAC, which can lyse the pathogenic bacterium Xanthomonas citri subsp. citri of citrus canker, providing a new biological control means for citrus canker.
[0008] The object of the present invention is achieved by the following technical solutions:
[0009] The present invention provides a citrus canker phage (Caudoviales phage) SAC, which is deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit number CGMCC No. 46373 and the deposit date of January 20, 2025.
[0010] The present invention provides the application of the above-mentioned citrus canker phage (Caudoviales phage) SAC in controlling plant diseases caused by Xanthomonas citri subsp. citri, and the plant disease is citrus canker.
[0011] The present invention also provides an agricultural fungicide, disinfectant or cleaner for controlling citrus canker caused by Xanthomonas citri subsp. citri.
[0012] Preferably, the agricultural fungicide, disinfectant or cleaner is a single preparation prepared from the citrus canker phage (Caudoviales phage) SAC or a compound preparation mainly composed of the citrus canker phage (Caudoviales phage) SAC.
[0013] Preferably, the dosage form of the agricultural fungicide, disinfectant or cleaner is a solution, powder or freeze-dried preparation.
[0014] The present invention also provides a method for controlling citrus canker caused by Xanthomonas citri subsp. citri, which is to spray the agricultural fungicide, disinfectant or cleaner on the citrus leaf surface or inject it into the plant body for controlling citrus canker.
[0015] The present invention also provides a method for preventing and controlling the contamination of Xanthomonas citri subsp. citri in the orchard environment, which is to spray the agricultural fungicide, disinfectant or cleaner on the environmental surface, and the environmental surface is the plant surface, planting soil, orchard air, surface water, and agricultural implements in the orchard.
[0016] Compared with the prior art, the present invention has the following beneficial technical effects:
[0017] The present invention provides a newly discovered citrus canker phage (Caudovirales phage) SAC, which can effectively lyse Xanthomonas citri subsp. citri, has good heat resistance and pH tolerance, is sensitive to high-intensity ultraviolet radiation, has good application prospects in the biological control of citrus canker, can be applied to pesticides, fungicides, disinfectants and cleaners, etc., can reduce the incidence of citrus canker, reduce the yield loss caused by diseases, and avoid the use of chemical pesticides at the same time, protecting the ecological environment and consumer health.
[0018] Description of Preservation Information
[0019] The citrus canker phage (Caudovirales phage) SAC has a preservation number of CGMCC No. 46373, a preservation date of January 20, 2025, and is preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, with the preservation address being the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing. Brief Description of the Drawings
[0020] The drawings included in the specification and constituting a part of the specification show the exemplary embodiments, features and aspects of the present invention together with the specification, and are used to explain the principles of the present invention.
[0021] Figure 1 shows the morphological characteristics of the citrus canker phage (Caudovirales phage) SAC;
[0022] Figure 2 shows the proteomic relationship and comparative genomic analysis results of the citrus canker phage (Caudovirales phage) SAC;
[0023] Figure 3 shows the phylogenetic tree of the terminase sequence of the citrus canker phage (Caudovirales phage) SAC;
[0024] Figure 4Results of FastINI analysis of phage (Caudoviales phage) SAC against citrus canker
[0025] Figure 5 Results of stability analysis and one-step growth curve analysis of phage (Caudoviales phage) SAC against citrus canker
[0026] Figure 6 Control effect of phage (Caudoviales phage) SAC against citrus canker Specific implementation mode
[0027] The present invention will be further described in detail below in conjunction with specific embodiments, which are explanations rather than limitations of the present invention.
[0028] The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified, and the experimental methods used in the following examples are conventional methods unless otherwise specified.
[0029] Example 1: Isolation, screening and identification of phage (Caudoviales phage) SAC against citrus canker
[0030] 1. Isolation and screening
[0031] Using Xanthomonas citri subsp. citri (Xcci) strain N8 as the starting strain. Take 5 g of soil sample in Long'an County, Nanning City and add it to 100 mL of bacterial liquid with OD 600 of 0.5 - 0.8, and culture overnight in a shaker at 28 °C and 220 r·min -1 . Centrifuge (4000×g, 15 min) to remove soil particles and bacteria, and then filter and sterilize with a sterile nylon filter membrane with a pore size of 0.22 μm to obtain a sterile suspension. First, inoculate 3 μL of the sample onto a double-layer plate containing Xcci N8 and culture it at 28 °C for 16 h to observe the formation of plaques on the plate. Then, gradient dilute the sterile suspension confirmed to contain phage, take 100 μL of the diluted sample, mix it with 900 μL of Xcci N8 bacterial liquid (OD 600 = 0.4 - 0.6), add it to 10 mL of NA semi-solid medium, and pour it onto a prepared NA plate, and culture it overnight in an incubator at 28 °C. Pick a single plaque and add it to 1 mL of SM buffer. Repeat this purification step 3 times to obtain a phage monoclonal sample, which is labeled as SAC.
[0032] 2. Morphological observation
[0033] SAC can form plaques with a diameter of approximately 2 mm on the double-layer plates of Xcci N8 strain. The formed plaques have a consistent morphology, presenting a regular circular shape, with a flat edge and clear boundaries, and are overall bright and transparent. Figure 1 -A). It has the characteristics of a virulent phage.
[0034] The suspension containing the phage was dropped on the copper grid. After incubation for 20 min, it was stained with 2% uranyl acetate for 5 min, and the excess stain was aspirated and dried at room temperature for 30 min. The morphology of the phage was observed using a transmission electron microscope (TEM). It was observed that the head diameter of SAC was approximately 60 nm, without a contractile tail sheath, and the tail was relatively short. Figure 1 -B). According to the morphological classification methods of Bradley and Ackermann, and combined with the latest virus classification criteria of the ICTV (International Committee on Taxonomy of Viruses), it can be determined that SAC belongs to the Podoviridae family under the order Caudovirales.
[0035] 3. Molecular biological identification
[0036] The whole genome of SAC was sequenced, and based on this, its terminal sequence was obtained. The nucleotide sequence of this terminal sequence is shown in SEQ ID NO.1.
[0037] By using BLASTn on the NCBI website to perform sequence alignment with the database, the results showed that SAC had the highest similarity with Xanthomonas phage CP2, with a comparison coverage rate of 82% and an identity of 98.02%. VipTree was used to establish a proteomic circular tree of SAC and 1003 phages. Figure 2 -A). The phages similar to SAC were selected to establish a proteomic rectangular tree. Figure 2 -B). SAC and Xanthomonas phage CP2 were on the same branch, and their genetic relationship was the closest. Genome collinearity analysis was performed with Xanthomonas phage CP2. Figure 2 -C). The results showed that there were two homologous fragments between SAC and Xanthomonas phage CP2, and the two homologous fragments had a relatively high similarity, but different positions.
[0038] The phage genome terminus is crucial for the entire DNA packaging process of the phage. The differences in phage species result in different terminal enzymes carried by them, and different types of terminal enzymes can further generate various types of genome terminal sequences. Therefore, we established a phylogenetic tree of the terminal enzyme sequences of phage SAC. Figure 3), SAC and Xanthomonas phage MET23-P3 are on the same branch with a score of 100. SAC and phage MET23-P3 have the same terminase sequence and are not on the same branch as Xanthomonas phage CP2. Based on the FastINI analysis results, the similarities between phage SAC and MET23-P3 and CP2 are 89.3618% and 94.247% respectively ( Figure 4 ), according to the latest classification rules of ICTV, if the genomic difference between two viruses is less than 95%, they are classified as different species. Therefore, phage SAC is a newly discovered species. This phage was named Caudoviales phage SAC for citrus canker. On January 20, 2025, this phage was sent to the General Microbiology Center of the China Committee for Culture Collection of Microorganisms for preservation, and the preservation number is CGMCC No. 46373.
[0039] Example 2 Biological characteristics of Caudoviales phage SAC for citrus canker
[0040] 1. Lysis spectrum of Caudoviales phage SAC for citrus canker
[0041] Using 17 strains of Xanthomonas pathogens preserved in the laboratory of the inventors as lysis targets, the lysis spectrum of the phage was measured by the spot method. This experimental design aims to evaluate the lysis ability of the selected phage against different Xanthomonas pathogens and study the lysis spectrum of Caudoviales phage SAC for citrus canker. It was found that 14 strains could be lysed by Caudoviales phage SAC for citrus canker, and these lysed strains were all different isolates of Xcci, while the 3 non-lysed strains were different species or pathovars of the genus Xanthomonas (Table 1). This result indicates that Caudoviales phage SAC for citrus canker has extremely strong specificity for Xanthomonas pathogens.
[0042] Table 1 Lysis ability of Caudoviales phage SAC for citrus canker against different Xanthomonas pathogens
[0043]
[0044] Note: +, sensitive to phage; -, insensitive to phage.
[0045] 2. Optimal multiplicity of infection of Caudoviales phage SAC for citrus canker
[0046] Adjust the concentration of Xcci N8 bacterial solution to 1×108 CFU·mL -1 According to the different multiplicity of infection (MOI = phage number / bacterial number), the corresponding amount of citrus canker phage (Caudoviales phage) SAC was added to the bacterial suspension and mixed. Incubate at 28 °C with shaking at 220 r·min -1 for 16 h, then centrifuge and collect the supernatant. Filter the supernatant using a 0.22 μm sterile nylon filter membrane, and determine the phage titer. The MOI value corresponding to the highest titer group is regarded as the optimal multiplicity of infection.
[0047] Analysis of the optimal multiplicity of infection showed that in the range of MOI values from 10 to 0.001, as the MOI value decreased, the titer of SAC gradually increased. When the MOI value was 0.001, the titer was the highest, reaching 4.5×10 11 PFU·mL -1 . Therefore, the optimal multiplicity of infection of citrus canker phage (Caudoviales phage) SAC is 0.001 (Table 2).
[0048] Table 2 Optimal multiplicity of infection of citrus canker phage (Caudoviales phage) SAC
[0049]
[0050] 3. Stability analysis of citrus canker phage (Caudoviales phage) SAC
[0051] After treating the suspension of citrus canker phage (Caudoviales phage) SAC at different temperatures for 60 min, the phage titer was determined by the double-layer plate method to measure its heat resistance.
[0052] Mix 100 μL of the suspension of citrus canker phage (Caudoviales phage) SAC with 900 μL of SM buffer with different pH values (pH 1.0 - 13.0), incubate at 28 °C for 60 min, and then determine the phage titer to measure its pH tolerance.
[0053] Pour 15 mL of the suspension of citrus canker phage (Caudoviales phage) SAC (1.0×1010 PFU·mL-1) into a sterile petri dish, place it 60 cm away from an ultraviolet lamp (power 18 W, light intensity 26.23 μW·cm-2) and irradiate continuously. Sample at predetermined time points and determine the titer to measure its resistance to ultraviolet radiation.
[0054] The results showed that during the temperature sensitivity test of the phage, the titer was relatively stable when the temperature was between 4 and 37 °C. After treatment at 50 °C for 60 min, the phage titer decreased slightly. After treatment at 60 °C for 60 min, the phage titer decreased significantly. After treatment at 70 °C for 60 min, the phage titer was zero and the phage was completely inactivated( Figure 5 -A); the titer was stable under the condition of pH 4 - 11; however, when the pH was 3.0 or 12.0, the titer of SAC was zero( Figure 5 -C); with the extension of the UV irradiation time, the titer of SAC continuously decreased. When the irradiation time was 5 min, the titer decreased by two orders of magnitude. When the irradiation time was 20 min, the titer decreased by 8 orders of magnitude. When the irradiation time was 25 min, the titer was zero and all the phages were inactivated( Figure 5 -B).
[0055] 4. One-step growth curve analysis of citrus canker phage (Caudoviales phage) SAC
[0056] According to the optimal multiplicity of infection, 100 μL of the suspension of citrus canker phage (Caudoviales phage) SAC was added to 10 mL of Xcci N8 in the logarithmic growth phase. Under the condition of 28 °C, it was adsorbed for 10 min, centrifuged at 12000×g for 1 min, and the precipitate was resuspended with NB medium. The resuspended culture solution was cultured in an incubator at 28 °C and 220 r·min-1. Samples were taken every 20 min, centrifuged at 12000×g for 1 min, and the phage titer in the supernatant was measured to draw the one-step growth curve of the phage
[0057] Through the one-step growth curve analysis of citrus canker phage (Caudoviales phage) SAC, it was found that during the period of 0 - 60 min after the phage infected the bacteria, the titer was relatively stable without obvious fluctuations, and this stage could be regarded as the latent period; within the time period of 60 - 140 min, the titer of SAC rapidly climbed and entered the lysis period; when the time exceeded 140 min, the titer tended to be stable, about 4.3×1011 PFU·mL-1, and entered the stationary period. The burst size of the phage was 430 PFU·cell-1( Figure 5 -D)
[0058] Example 3 Control effect of citrus canker phage (Caudoviales phage) SAC
[0059] 1. Control effect of the phage in liquid medium
[0060] The Xcci N8 bacterial solution (OD600 = 0.5 - 0.8) was aliquoted into conical flasks and divided into two groups. No phage was added to the conical flasks in the first group, and the suspension of citrus canker phage (Caudoviales phage) SAC was added to the conical flasks in the second group at the optimal multiplicity of infection. These two groups of conical flasks were co-cultured in a shaker at 28 °C and 220 r·min-1, and the absorbance was measured every 1 h.
[0061] The results showed that after the addition of citrus canker phage (Caudoviales phage) SAC, the absorbance of the bacterial solution continued to increase in a short time, but the increase amplitude was less than that of the control group. At the 6th h, the absorbance of the bacterial solution began to decrease, and since the 8th h, the absorbance of the bacterial solution has remained at a low level ( Figure 6 -A).
[0062] 2. Control effect of phage in plants
[0063] Using Xcci N8 as the standard strain, it was inoculated into citrus leaves by the pressure infiltration method. The citrus leaf treatments were divided into two groups. In the first group, only Xcci N8 was inoculated without any other treatment. In the second group, while inoculating Xcci N8, the suspension of citrus canker phage (Caudoviales phage) SAC was sprayed onto the citrus leaves. Thereafter, the same suspension was sprayed every two days. When sampling, leaves with the same area should be cut at the position of the lesions, and the CFU of Xcci per unit area at the cut leaves was measured. Sampling should be carried out before spraying the phage.
[0064] The results showed that from the 3rd to the 9th day of the experiment, the growth of Xcci in the plants was slow, much less than that of the Xanthomonas control group. After the 9th day, Xcci N8 no longer grew and its CFU began to decrease ( Figure 6 -B), and the size of the lesions in the control group was significantly larger than that in the citrus canker phage (Caudoviales phage) SAC treatment group ( Figure 6 -C). It indicates that citrus canker phage (Caudoviales phage) SAC can effectively inhibit the infection of Xcci on citrus plants.
[0065] The foregoing description of the specific exemplary embodiments of the present invention is for purposes of illustration and exemplification. These descriptions are not intended to limit the present invention to the precise forms disclosed, and it is obvious that many changes and variations can be made in light of the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical applications, so that those skilled in the art can implement and utilize various different exemplary embodiments of the present invention as well as various different selections and changes. The scope of the present invention is intended to be defined by the claims and their equivalents.
Claims
1. A citrus canker phage (Caudovialesphage) SAC, characterized in that: The bacteriophage is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number being CGMCC No.46373 and the deposit date being January 20, 2025.
2. The use of the citrus canker phage (Caudovialesphage) SAC according to claim 1 in preventing and controlling plant diseases caused by Xanthomonas citri subsp. citrus, characterized in that: The plant disease is citrus canker.
3. An agricultural fungicide, disinfectant or cleaner for preventing and treating citrus canker caused by Xanthomonas citri subsp. citri, characterized in that: The agricultural fungicide, disinfectant or cleaning agent is a single preparation prepared with citrus canker phage (Caudovialesphage) SAC or a compound preparation prepared with citrus canker phage (Caudovialesphage) SAC as a main component.
4. The agricultural fungicide, disinfectant or cleaner according to claim 3, characterized in that: The dosage form of the agricultural fungicide, disinfectant or cleaner is solution, powder or freeze-dried agent.
5. A method for preventing and treating citrus canker caused by Xanthomonas citri subspecies citrus, comprising spraying the agricultural fungicide, disinfectant or detergent according to claim 3 or claim 4 on the citrus leaves or injecting it into the plant body.
6. A method for preventing and controlling Xanthomonas citri subsp. citri contamination in an orchard environment, comprising spraying the agricultural fungicide, disinfectant or detergent according to claim 3 or claim 4 on the surface of the environment.
7. The method according to claim 6, characterized in that: The environmental surfaces are plant surfaces, planting soil, orchard air, surface water, and agricultural tools in the orchard.