Carpet grass xanthomonas phage and application thereof

Abstract

The invention provides a carpet grass xanthomonas bacteriophage XAC18P1 capable of preventing and treating citrus canker and application thereof, and belongs to the technical field of biology, the bacteriophage is preserved in China Center for Type Culture Collection on May 24, 2024, the preservation address is No. 299 on eight road, Wuchang District, Wuhan City, Hubei Province, and the preservation number is CCTCC NO: M 20241060. The bacteriophage XAC18P1 provided by the invention is wide in host range, the splitting rate is as high as 100%, and the bacteriophage XAC18P1 has relatively strong bactericidal ability and can be used for prevention and control and treatment of citrus canker, so that the incidence rate of citrus seedlings is reduced by 90% or above. In addition, the bacteriophage is high in fermentation rate and strong in stability, and has a good commercial prospect.

Description

Technical Field

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

[0002] Citrus bacterial canker originated in India in South Asia and has since spread to Asia, South Africa, Central Africa, the Middle East, Australia, New Zealand, the Pacific Islands, South America and North America. The disease is caused by Xanthomonas axonopodispv. citri and is currently the most destructive bacterial disease in the citrus industry. It can harm the leaves, branches and fruits of citrus. In severe cases, it can cause a large number of leaves to fall, greatly affecting the photosynthesis of the plant, leading to the weakening of the tree, and significantly reducing the yield and quality of citrus fruit. Not only do the diseased fruits look ugly, but the sugar, vitamin C and pectin content they contain will also be reduced, which will lead to a low market price and cause serious losses.

[0003] Traditionally, the prevention and control of Xanthomonas aeruginosa has mostly used chemical therapies including antibiotics, such as Bordeaux mixture, cuprous oxide, and kasugamycin, or through pruning, windbreak planting, and other methods. Although chemical therapies are fast and effective, the frequent use of antibiotics will cause pathogens to develop drug resistance. Pruning branches and leaves, planting windbreaks, and other methods can only prevent to a certain extent, and the effect is limited. As a potential natural, green, safe and effective alternative to antibiotics, bacteriophages have great advantages in their high host specificity. Screening out suitable bacteriophages for Xanthomonas aeruginosa is of great significance for the prevention and control of citrus canker. Summary of the invention

[0004] In view of this, the present invention proposes a broad-spectrum, high-fermentation-rate Xanthomonas phage of carpet grass and application thereof, which is of great significance for preventing and treating citrus canker.

[0005] The first aspect of the present invention provides a bacteriophage, which was deposited in the China Center for Type Culture Collection on May 24, 2024, with the deposit address being No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number being CCTCC NO: M 20241060.

[0006] Bacteriophage XAC18P1 can efficiently lyse Xanthomonas carpetgrass from various sources, has a wide host range, a lysis rate of 100%, and exhibits excellent bactericidal ability. It can effectively inhibit the growth of Xanthomonas carpetgrass within 36 hours and can be used for the prevention, control and treatment of citrus canker. At the same time, in the prevention and control experiment, bacteriophage XAC18P1 also showed excellent prevention and treatment effects, which can reduce the incidence of citrus seedlings by more than 90%. In addition, the phage has a high fermentation rate and can produce 1.7×10 11 PFU / ml progeny, which is very conducive to the preparation of high-concentration phage preparations. At the same time, the phage is also extremely stable at 4-45°C and pH 4-10, and has good application potential.

[0007] The second aspect of the present invention provides a bacterial suspension, wherein the bacterial suspension comprises the bacteriophage described in the first aspect.

[0008] The third aspect of the present invention provides use of the bacteriophage described in the first aspect and the bacterial suspension described in the second aspect in the preparation of pesticides.

[0009] The fourth aspect of the present invention provides a pesticide, which contains at least one of the bacteriophage described in the first aspect and the bacterial suspension described in the second aspect.

[0010] According to a specific embodiment of the present invention, the pesticide further includes a pesticide adjuvant.

[0011] According to a specific embodiment of the present invention, the pesticide adjuvant includes at least one of a filler and a carrier, an emulsifier, a dispersant, a wetting agent, a spreader, a solvent, a sticker, a stabilizer, a synergist, and a spray adjuvant.

[0012] According to a specific embodiment of the present invention, the formulation of the pesticide includes any one of powder, wettable powder, emulsion, emulsifiable concentrate, granule, microgranule and oil.

[0013] The fifth aspect of the present invention provides use of the bacteriophage described in the first aspect, the bacterial suspension described in the second aspect, and the pesticide described in the fourth aspect in preventing or treating citrus canker.

[0014] According to a specific embodiment of the present invention, the citrus canker is caused by Xanthomonas aegypti and / or Xanthomonas aegypti pv. citrus.

[0015] The phage screened by the present invention and a series of preparations prepared by the phage of the present invention have the following beneficial effects compared with the prior art:

[0016] (1) The bacteriophage has a broad spectrum of killing Xanthomonas spp. and has an extremely high lysis rate. The lysis rate of 15 strains of Xanthomonas spp. citrus pathogens was 100%;

[0017] (2) The phage has a high fermentation rate and can produce 1.7×10 11 PFU / ml progeny, which is conducive to the preparation of high-concentration phage preparations;

[0018] (3) The bacteriophage is extremely stable, still has a high titer at 4-45°C and pH 4-10, and is resistant to storage and transportation;

[0019] (4) The phage shown in Figure 4 has an excellent control effect on Xanthomonas aeruginosa and can reduce the incidence of citrus seedlings by 90%.

[0020] Collection information:

[0021] Strain name: Xanthomonas phage XAC18P1

[0022] Storage date: May 24, 2024

[0023] Depository: China Center for Type Culture Collection (CCTCC)

[0024] Deposit number: CCTCC NO:M 20241060 BRIEF DESCRIPTION OF THE DRAWINGS

[0025] 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 or the description of the prior art 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.

[0026] Figure 1 This is a plaque picture of bacteriophage XAC18P1 in Example 1 of the present invention;

[0027] Figure 2 is a phylogenetic map of bacteriophage XAC18P1 in Example 1 of the present invention;

[0028] Figure 3 is the titer of bacteriophage XAC18P1 at different infection multiplicities in Example 2 of the present invention;

[0029] Figure 4 This is a temperature stability diagram of bacteriophage XAC18P1 in Example 4 of the present invention;

[0030] Figure 5 This is the acid-base stability diagram of bacteriophage XAC18P1 in Example 5 of the present invention;

[0031] Figure 6This is a graph showing the bactericidal ability of bacteriophage XAC18P1 against Xanthomonas aeruginosa XAC18 in Example 6 of the present invention. DETAILED DESCRIPTION

[0032] Embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0033] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0034] In this document, the terms “contain”, “include” or “comprise” are open expressions, that is, they include the contents specified in the present invention but do not exclude other contents.

[0035] As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0036] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention provides drawings, which are part of the disclosure of the present invention. They are mainly used to illustrate the embodiments and can be used in conjunction with the relevant descriptions in the specification to explain the operating principles of the embodiments. By referring to these contents, ordinary technicians in this field should be able to understand other possible implementation methods and advantages of the present invention.

[0037] In the following example,

[0038] The formula of NB liquid culture medium is: peptone 10g, beef extract powder 3g, sodium chloride 5g, purified water 1000mL, pH 7.2±0.2, and it is used after being sterilized by pressure steam at 121℃.

[0039] The formula of NB semi-solid medium is: peptone 10g, beef extract powder 3g, sodium chloride 5g, agar 7.5g, purified water 1000mL, pH 7.2±0.2, and it is used after being sterilized by pressure steam at 121℃.

[0040] The formula of NA plate medium is: peptone 10g, beef extract powder 3g, sodium chloride 5g, agar 15g, purified water 1000mL, pH 7.2±0.2, and it is used after being sterilized by pressure steam at 121℃.

[0041] The formula of SM buffer is: 2.46 g of magnesium sulfate heptahydrate, 5.84 g of sodium chloride, 0.1 g of gelatin, 7.88 g of tris(hydroxymethyl)aminomethane hydrochloride, and 1000 mL of purified water, and it is used after being sterilized by pressure steam at 121°C.

[0042] In the embodiment, the bacteriophage with biological activity is the Xanthomonas agarica phage XAC18P1, and the host strain is the Xanthomonas agarica pv. citri citri XAC18.

[0043] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] Example 1: Isolation and purification of Xanthomonas phage XAC18P1

[0045] Soil from a citrus orchard where citrus canker disease occurred was collected, and 5 g of soil sample was mixed with 15 ml of Xanthomonas aeruginosa XAC18 (isolated from a soil sample from a citrus orchard in Yichang, Hubei Province) in the middle logarithmic growth period, and placed on a constant temperature shaker at 28°C and 200 r / min for 24 hours to enrich bacteriophages. The enriched solution was centrifuged at 6000×g for 8 minutes, and the supernatant was taken and sterilized by filtering with a 0.22um microporous filter membrane to obtain a filtrate containing bacteriophages. The filtrate was diluted gradiently, and 100 uL of the dilution was mixed evenly with 250 uL of its host Xanthomonas aeruginosa XAC18, and allowed to stand for 15 minutes to allow it to fully bind to the receptors on the bacterial surface. The above mixture was added to 7 mL of NB semi-solid agar medium cooled to 50°C, and immediately spread on a solidified nutrient agar plate and mixed evenly. After the semi-solid agar medium solidified, it was inverted and cultured at 28°C for 24 hours to observe the growth of phage plaques. On the double-layer plate where plaques are formed, pick up a large and transparent single plaque with a sterile pipette tip, inoculate it in 3mL NB liquid culture medium, add 200uL carpet grass Xanthomonas XAC18 liquid and mix well, culture overnight at 28℃ and 200rpm, centrifuge at 6000×g for 8min, take the supernatant and filter it through a 0.22μm microporous membrane, and observe the plaque morphology by double-layer plate method. After repeating the operation 3 times, the isolation and purification of a single phage strain can be completed.

[0046] The isolated and purified phage produced circular plaques on Xanthomonas carpetgrass lawn (e.g. Figure 1 As shown), the diameter is about 18 mm. The phage was named phage XAC18P1 and sent to Annouda Gene Technology Co., Ltd. for whole genome sequencing.

[0047] The measured sequences were aligned by Blastn on NCBI, and a phylogenetic tree was constructed based on the alignment results. Figure 2 As shown, XAC18P1 is a species of the Pradovirus genus, and its closest relationship to BUCT603 is 86%, and it is determined to be a newly discovered phage strain.

[0048] The phage XAC18P1 was sent to the China Center for Type Culture Collection for preservation on May 24, 2024, and the classification name was: Xanthomonas axonopodis phage XAC18P1, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, preservation number: CCTCC NO: M 20241060.

[0049] Example 2: Determination of the optimal multiplicity of infection of Xanthomonas aegypti phage XAC18P1

[0050] The host Xanthomonas carpetgrass XAC18 colony was picked and placed in 3 mL NB liquid culture medium, and cultured at 200 rpm in a shaker at 28°C for 24 hours to obtain a host bacterial suspension. The bacterial suspension was transferred to 10 mL NB culture medium at a ratio of 1:100, and cultured at 28°C and 200 rpm to the early logarithmic phase. An equal volume of 5 tubes of 10-fold dilutions of phage were added to the host bacterial solution to achieve different infection multiplicities (MOI: phage / bacteria is 10, 1, 0.1, 0.01, 0.001). After the culture was completed, the fermentation liquid was centrifuged at 6000×g for 8 minutes and the supernatant was collected. The supernatant was filtered through a 0.22μm microporous filter and counted by double-layer plate method. Each point was cultured in duplicate tubes and the average value was taken. The condition that produced the highest phage titer was the optimal infection multiplicity.

[0051] The results are as follows Figure 3 As shown in the figure, when MOI = 0.1, 1.7×10 11 PFU / mL of progeny has a high fermentation rate, which is beneficial to the preparation of phage fermentation broth.

[0052] Example 3: Host range determination of bacteriophage XAC18P1

[0053] In order to understand the host range of bacteriophage XAC18P1, this study used 18 strains of Xanthomonas citri var. citri from different locations to test the host range of bacteriophage XAC18P1, and the test was determined by the spot method. Prepare the test strains, inoculate them in centrifuge tubes containing 3mL NB liquid culture medium, and culture them at 28℃ and 200rpm for 24h to obtain bacterial suspensions of each strain. Take 400μL of bacterial suspension and mix it with NB semi-solid culture medium and spread it on the prepared nutrient agar plate. After solidification, drop 5uL of the titer is about 1×10 9 PFU / mL of bacteriophage XAC18P1. After drying, place it at 28℃ for 24h and observe the results. Each point was done twice to ensure its accuracy. The results are shown in Table 1. Bacteriophage XAC18P1 can lyse all 18 tested Xanthomonas citri var. citri, with a lysis rate of 100%, indicating that bacteriophage XAC18P1 has a wide host range and good application potential.

[0054] Table 1 Host range of Xanthomonas phage XAC18P1

[0055]

[0056]

[0057] +: plaques present; -: no plaques present

[0058] Example 4: Temperature stability test of bacteriophage XAC18P1

[0059] The phage suspension (~1×10 10 PFU / ml) was dispensed into 8 1.5ml microcentrifuge tubes and placed at different temperatures (4℃, 25℃, 37℃, 45℃, 55℃, 65℃, 75℃). After 1 hour, the titer was determined by double-layer agar plate method. Each group was tested in duplicate and the average value was taken. Figure 4 As shown, the activity of Xanthomonas phage XAC18P1 of carpet grass changed little in the range of 4℃ to 45℃. Although the survival rate decreased at 55℃, it still had a high titer.

[0060] Example 5: Acid-base stability test of bacteriophage XAC18P1

[0061] SM buffer was adjusted to 5 different pH values ​​(2, 4, 6, 8, 10) using 10N sodium hydroxide (NaOH) and 6N hydrochloric acid (HCl). The phage suspension was diluted in a 1.5 ml microcentrifuge tube with the pH adjusted SM buffer (final concentration was about 1×10 10 PFU / ml)

[0062] Place at room temperature, dilute after 1 hour and measure the titer using double agar plate method. Duplicate tubes were used for each group and the average value was taken. Figure 5 As shown, the activity of Xanthomonas phage XAC18P1 varied slightly between pH = 4 and pH = 10.

[0063] Example 6: In vitro bactericidal ability test of bacteriophage XAC18P1

[0064] XAC18 was cultured in 200 ml NB liquid medium and diluted to OD 600 0.1(~1.1×10 8 CFU / ml), 2.2×10 9 The Xanthomonas phage XAC18P1 was added to the host bacterial solution with a volume of 100 μl PFU (MOI of 0.1), and the control group was added with an equal volume of SM buffer. The culture was cultured at 28°C and 200 rpm for 36 hours, and the OD value of the culture solution was tested at intervals of 1 hour. 600 This experiment was repeated in duplicate to calculate the average value and plot the growth curve of the strain. Figure 6 As shown in the figure, phage XAC18P1 can effectively inhibit the growth of Xanthomonas sphaeroides XAC18. After 36 hours of co-culture, the OD of XAC18 bacterial solution was 600 The value was only 0.263, compared with 1.17 in the control group, whose growth was obviously inhibited by bacteriophage XAC18P1, indicating that bacteriophage XAC18P1 has good in vitro bactericidal ability.

[0065] Example 7: Determination of control effect

[0066] 90 citrus seedlings were selected for planting in the laboratory and divided into a control group, an infection group, and a phage treatment group, with 3 parallels in each group and 10 citrus seedlings in each parallel. The temperature was controlled at 25℃ from 08:00 to 18:00 and 20℃ from 18:00 to 08:00; full-spectrum plant growth lights were used from 06:00 to 18:00 every day, and the light intensity was maintained at 200-400 micromoles / square meter / second; the air humidity was maintained at 60-70% and good air circulation was maintained; watering was done twice a day to keep the soil moist. The growth of the citrus seedlings was observed every day, and the incidence of citrus seedlings in each group was recorded.

[0067] The host bacteria of the phage of the present invention were cultured to reduce the concentration of Xanthomonas spp. XAC18 to about 1.1×10 8 CFU / ml. Ferment and dilute the bacteriophage XAC18P1 of the present invention to about 1.1×10 7PFU / ml. 90 citrus seedlings were treated with the above bacterial solution and phage solution. The control group used PBS with equal volume of bacterial solution and SM buffer with equal volume of phage solution, and the infection group used equal volume of SM buffer. The results are shown in Table 2. The control group had diseased citrus seedlings. In the infection group, 14 citrus seedlings were infected at 5 days, with an incidence rate of 46.67%; 28 citrus seedlings were infected 10 days later, with an incidence rate as high as 93.33%. In the phage treatment group, 1 citrus seedling was infected at 5 days, with an incidence rate of 3.33%; 1 citrus seedling was infected 10 days later, with an incidence rate of only 3.33%. In the phage pretreatment group, 0 citrus seedlings were infected at 5 days; 0 citrus seedlings were infected 10 days later, and no citrus seedlings were infected, with an incidence rate of 0%. By comparison, it was found that the phage XAC18P1 reduced the incidence of citrus seedlings by more than 90% regardless of prior prevention or post-infection treatment. It can be seen that in practical application, bacteriophage XAC18P1 can significantly reduce the incidence of citrus seedlings, thereby reducing the harm caused by Xanthomonas aeruginosa in crop cultivation.

[0068] Table 2 Determination of the control effect of bacteriophage XAC18P1 on Xanthomonas spp.

[0069]

[0070] Based on Examples 1-7, it can be seen that the bacteriophage XAC18P1 provided by the present invention can efficiently lyse Xanthomonas carpetgrass from various sources, has a wide host range, a lysis rate of 100%, and exhibits excellent bactericidal ability. It can effectively inhibit the growth of Xanthomonas carpetgrass within 36 hours and can be used for the prevention, control and treatment of citrus canker. At the same time, in the prevention and control experiment, the bacteriophage XAC18P1 also showed excellent prevention and treatment effects, which can reduce the incidence of citrus seedlings by more than 90%. In addition, the phage has a high fermentation rate and can produce 1.7×10 11 PFU / ml progeny, which is very conducive to the preparation of high-concentration phage preparations. At the same time, the phage is also extremely stable in an environment of 4°C to 45°C and pH=4 to pH=10, and has good application potential.

[0071] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A bacteriophage, characterized in that: The deposit number of the bacteriophage is CCTCC NO: M20241060.

2. A bacterial suspension, characterized in that: The bacterial suspension contains the bacteriophage according to claim 1.

3. Use of the bacteriophage according to claim 1 and the bacterial suspension according to claim 2 in the preparation of pesticides.

4. A pesticide, characterized in that: The pesticide contains at least one of the bacteriophage according to claim 1 and the bacterial suspension according to claim 2.

5. The pesticide according to claim 4, characterized in that The pesticide includes a pesticide adjuvant.

6. The pesticide according to claim 5, characterized in that The pesticide adjuvant includes at least one of a filler and a carrier, an emulsifier, a dispersant, a wetting agent, a spreader, a solvent, a sticker, a stabilizer, a synergist, and a spray adjuvant.

7. The pesticide according to claim 4, characterized in that The formulation of the pesticide includes any one of powder, wettable powder, emulsion, emulsifiable concentrate, granule, microgranule and oil.

8. Use of the bacteriophage according to claim 1, the bacterial suspension according to claim 2, and the pesticide according to any one of claims 4 to 7 in preventing or treating citrus canker.

9. The use according to claim 8, characterized in that The citrus canker is caused by Xanthomonas aegypti and / or Xanthomonas aegypti pv. citrus.

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