Bacteriophage for preventing and controlling ginger stem rot and use thereof

Abstract

Provided are a bacteriophage of Achromobacter xylosoxidans for preventing and controlling ginger stem rot, and the use thereof. The use method comprises: step 1, preparing a strain; step 2, preparing a culture medium and a reagent; step 3, preparing a bacteriophage suspension; step 4, performing an activity assay and storing; step 5, breeding and transplanting; and step 6, inoculating with the bacteriophage. By means of preventing and controlling ginger stem rot using the bacteriophage of Achromobacter xylosoxidans, the problem of drug resistance of pathogenic bacteria on a bactericidal preparation is avoided. In addition, prevention and control using the bacteriophage is milder and more effective than physical sterilization means.

Description

Bacteriophage for preventing and treating ginger stem base rot disease and application thereof Technical Field

[0001] The present invention relates to the technical field of ginger disease prevention and control, and in particular to a bacteriophage for preventing and controlling ginger stem base rot disease and an application thereof. Background Art

[0002] Ginger is a perennial herb of the genus Zingiberaceae, native to the tropical regions of Southeast Asia. It prefers a warm and humid climate and has weak cold and drought resistance. It is a plant with both edible and medicinal value. Its unique spicy aroma and medicinal effects make it widely used in daily life. However, ginger stem base rot disease may occur during the cultivation and planting process. Among them, the stem base rot caused by Achromobacter xylosoxidans is extremely harmful to ginger. The stem base of the diseased plant becomes soft, brown and water-soaked, and the rhizome shows symptoms of browning and water-soaked rot.

[0003] Existing methods for preventing and controlling ginger stem rot have the following defects: First, with the extensive and long-term use of antibiotic bactericidal preparations, Achromobacter xylosoxidans is prone to develop drug resistance, and the drug resistance becomes stronger over time, reducing the effectiveness of disease prevention and control; Second, because chemical agents have a broad spectrum and poor target targeting, they will affect the survival of normal and beneficial microorganisms while killing Achromobacter xylosoxidans, destroy the soil ecological environment, and affect the growth of ginger; Third, the use of physical means to kill Achromobacter xylosoxidans in high-temperature greenhouses will not only kill Achromobacter xylosoxidans, but also cause stress or even death to other microorganisms that are intolerant to high temperatures. Field management methods such as clearing the garden and crop rotation cannot completely eliminate pathogens, and only treat the symptoms but not the root cause.

[0004] Summary of the Invention

[0005] The object of the present invention is to provide a bacteriophage for preventing and treating ginger stem base rot disease and its application, so as to solve the problems raised in the above background technology.

[0006] To achieve the above objectives, the present invention provides the following technical solutions: a bacteriophage for preventing and controlling ginger stem base rot disease, comprising: a xylose-oxidizing Achromobacter phage, wherein the xylose-oxidizing Achromobacter phage is one or more of phiAxp-1, phiAxp-2 and phiAxp-3.

[0007] A bacteriophage application for preventing and controlling ginger stem base rot disease, comprising the steps of: 1. strain preparation; 2. culture medium and reagent preparation; 3. phage suspension preparation; 4. activity determination and preservation; 5. breeding and transplanting; and 6. phage inoculation.

[0008] In the above step 1, the pathogenic bacteria Achromobacter xylosoxidans and bacteriophage are prepared;

[0009] In the above step 2, the culture medium and related reagents required for preparing the bacterial solution are prepared;

[0010] In the above step 3, a phage suspension is prepared and enriched;

[0011] In the above step 4, the activity of the phage is determined and the phage is preserved;

[0012] In the above step 5, ginger is bred and transplanted;

[0013] In the above step 6, the phage inoculation is carried out by rhizome injection method.

[0014] As a further technical solution of the present invention, in the step one, Achromobacter xylosoxidans is isolated from the rhizosphere of severely diseased ginger, and a strongly lytic phage is obtained from its rhizosphere soil by screening through a double-layer plate method, and one or more phages of phiAxp-1, phiAxp-2 and phiAxp-3 are prepared for co-culture, and the phage suspension is placed in a glycerol tube and stored in an ultra-low temperature freezer at -80°C.

[0015] As a further technical solution of the present invention, in the step 2, NA liquid culture medium is prepared, the ingredients of which are 5 grams of peptone, 3 grams of beef extract, 10 grams of glucose and 800 milliliters of pure water, the pH is adjusted to 7.2-7.4, and the medium is sterilized by autoclave at 120°C for 45 minutes. NA solid culture medium, the ingredients of which are 5 grams of peptone, 3 grams of beef extract, 10 grams of glucose, 800 milliliters of pure water and 25 grams of agar, the pH is adjusted to 7.2-7.4, and the medium is sterilized by autoclave at 120°C for 45 minutes.

[0016] As a further technical solution of the present invention, in step 2, the selective culture medium is added sequentially to the NA liquid culture medium with 50 mg of crystal violet, 25 mg of bacitracin, 5 mg of penicillin, 50 mg of bacitracin, and 100 mg of polybactin; the buffer is added with 2 g of magnesium sulfate, 5 g of sodium chloride, and 5 g of gelatin, 50 ml of the buffer base liquid, and pure water is added to make the volume to 1000 ml, and the mixture is sterilized under high pressure at 120°C for 30 minutes.

[0017] As a further technical solution of the present invention, in step three, the pathogenic bacteria Achromobacter xylosoxidans is inoculated and cultured on a selective culture medium and cultured in a constant temperature incubator at 32°C for 36 hours. High-quality colonies are selected and transferred to NA liquid culture medium and placed in a shaker device for shaking and culture for 48 hours.

[0018] As a further technical solution of the present invention, in the step three, the phage suspension is taken out, 15 ml of the xylose oxidizing colorless bacteria liquid is taken and placed in the NA solid culture medium and mixed, and then poured onto the culture medium plate, the phage suspension is spotted on the plate, and cultured at a constant temperature of 32°C for 24 to 36 hours, during which time the phage plaques are selected and transferred to the xylose oxidizing colorless bacteria liquid, shaken and allowed to stand for 15 minutes, and cultured at a constant temperature of 32°C for 12 to 15 hours to allow the phage to fully invade the xylose oxidizing colorless bacteria for lysis and proliferation. The completely lysed culture medium contains a large amount of bacterial fragments in the form of limited filaments. The suspension is placed in a centrifuge at 12,000 rpm and centrifuged for 10 minutes. The supernatant is collected and filtered through a filter membrane to obtain a phage suspension.

[0019] As a further technical solution of the present invention, in step 4, the quantity and activity of the phage suspension are measured, the concentration is diluted with a buffer solution, and the phage powder is prepared and stored at ultra-low temperature.

[0020] As a further technical solution of the present invention, in step five, ginger seedlings are selected for breeding, and then the ginger seedlings are transplanted into transplanting trays, and the soil matrix attached to the rhizomes is washed off during transplanting.

[0021] As a further technical solution of the present invention, in step six, the phage powder is prepared into a suspension with a buffer solution, and then 75% alcohol is sprayed on the rhizome of ginger using a spray gun for disinfection. The phage is injected and inoculated into the rhizome of ginger using a syringe. The inoculated ginger seedlings are covered with soil and cultivated for one week before being transferred to a greenhouse area. The phage suspension can be appropriately irrigated at the roots of the transplanting pit.

[0022] Compared with the prior art, the present invention has the following beneficial effects: the present invention prevents and controls the stem base rot disease of ginger by using the Achromobacter xylosoxidans phage, thereby avoiding the problem of pathogenic bacteria developing resistance to bactericidal preparations, and the prevention and control effect is more stable. The phage has strong targeting, and can kill the Achromobacter xylosoxidans without affecting the survival of normal and beneficial microorganisms, thereby maintaining the soil flora structure and community diversity, and being more beneficial to ginger growth. At the same time, the phage prevention and control is more gentle and effective than physical sterilization methods, and can prevent and control the stem base rot disease caused by Achromobacter xylosoxidans for a long time, with a low recurrence rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG1 is a flow chart of the application method of the present invention. DETAILED DESCRIPTION

[0024] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. 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 making creative efforts are within the scope of protection of the present invention.

[0025] Please refer to Figure 1, an embodiment of the present invention provides: a phage for preventing and treating ginger stem base rot disease, including: a xylosoxidans Achromobacter phage, and the xylosoxidans Achromobacter phage is one or more of phiAxp-1, phiAxp-2 and phiAxp-3.

[0026] A bacteriophage application for preventing and controlling ginger stem base rot disease, comprising the steps of: 1. strain preparation; 2. culture medium and reagent preparation; 3. phage suspension preparation; 4. activity determination and preservation; 5. breeding and transplanting; and 6. phage inoculation.

[0027] In the above step 1, Achromobacter xylosoxidans is isolated from severely diseased ginger stem tissue, and a strongly lytic phage is screened from its rhizosphere soil by a double-layer plate method, and one or more phages of phiAxp-1, phiAxp-2, and phiAxp-3 are prepared for co-culture, and the phage suspension is placed in a glycerol tube and stored in an ultra-low temperature freezer at -80°C;

[0028] Wherein in the above step 2, prepare the culture medium and related reagents required for preparing the bacterial solution, prepare NA liquid medium, the components are 5 grams of peptone, 3 grams of beef extract, 10 grams of glucose and 800 milliliters of pure water, adjust the pH to 7.2-7.4, and autoclave at 120°C for 45 minutes, NA solid medium, the components are 5 grams of peptone, 3 grams of beef extract, 10 grams of glucose, 800 milliliters of pure water and 25 grams of agar, adjust the pH to 7.2-7.4, and autoclave at 120°C for 45 minutes, selective culture medium, add 50 mg of crystal violet, 25 mg of bacitracin, 5 mg of penicillin, 50 mg of bacitracin and 100 mg of polybactin in sequence to the NA liquid medium, buffer, 2 grams of magnesium sulfate, 5 grams of sodium chloride and 5 grams of gelatin, add 50 milliliters of buffer base solution, add pure water to 1000 milliliters, and autoclave at 120°C for 30 minutes;

[0029] wherein in the above step three, a phage suspension is prepared and enriched, the pathogenic bacteria of colorless xylose oxidizing bacteria is inoculated and cultivated on a selective culture medium, cultured in a constant temperature incubator at 32°C for 36 hours, high-quality colonies are selected and transferred to NA liquid culture medium, placed in a shaking table device for shaking and culture for 48 hours, the phage suspension is taken out, 15 ml of colorless xylose oxidizing bacteria liquid is put into NA solid culture medium and mixed, then poured on a culture medium plate, the phage suspension is spotted on the plate, and cultured at 32°C for 24 to 36 hours, during which time plaques are selected and transferred to the colorless xylose oxidizing bacteria liquid, shaken and allowed to stand for 15 minutes, and cultured at 32°C for 12 to 15 hours to allow the phage to fully invade the colorless xylose oxidizing bacteria for lysis and proliferation. The completely lysed culture solution contains a large amount of bacterial fragments in the form of limited filaments. The suspension is placed in a centrifuge at 12,000 rpm for 10 minutes, and the supernatant is collected and filtered through a filter membrane to obtain a phage suspension;

[0030] In the above step 4, the number and activity of the phage suspension are determined, the concentration is diluted with a buffer solution, and the phage powder is prepared and stored at ultra-low temperature;

[0031] In the above step 5, ginger seedlings are selected for breeding, and then the ginger seedlings are transplanted into transplanting trays, and the soil matrix attached to the rhizomes is washed off during transplanting;

[0032] In the above step six, the phage powder is prepared into a suspension with a buffer solution, and then 75% alcohol is sprayed on the rhizome of ginger using a spray gun for disinfection. The phage is injected and inoculated into the rhizome of ginger using a syringe. The inoculated ginger seedlings are covered with soil and cultivated for one week before being transferred to the greenhouse area. The phage suspension can be appropriately irrigated at the roots of the transplanting pit.

[0033] Based on the above, the advantages of the present invention are: the present invention uses the xylose-oxidizing Achromobacter phage to prevent and treat ginger stem base rot, avoiding the problem of pathogenic bacteria developing resistance to bactericidal preparations, with efficient lysis and high potency. For soil-borne diseases, specific phages can efficiently and quickly attach to bacterial cells and inject genetic material into host cells, causing cell lysis and death, and co-evolving with pathogens. When the phage invades the bacterial cell, the bacterial cell must evolve into a stronger bacterium in order to survive, and the phage must also evolve into a phage with stronger infection ability in order to obtain sufficient material resources. Thus, it forms a co-evolution with the host cells. Therefore, it can overcome the resistance evolution of bacteria to a certain extent, and the treatment effect is more stable, and it has strong targeting and special infection. The phage only specifically targets one kind of bacteria. Therefore, while infecting specific microorganisms, it will not bring any impact on the beneficial bacteria in the soil, maintaining the structure and diversity of the soil flora. At the same time, phage control is more gentle and effective than physical sterilization methods, and can prevent and control the stem base rot disease caused by Achromobacter xylosoxidans for a long time, with a low recurrence rate.

[0034] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims, not the foregoing description, and all variations within the meaning and range of equivalents of the claims are intended to be included therein. Any reference sign in a claim should not be construed as limiting the claim to which it relates.

Claims

1. A bacteriophage for preventing and treating ginger stem base rot disease, comprising: The xylose-oxidizing Achromobacter phage is characterized in that the xylose-oxidizing Achromobacter phage is one or more of phiAxp-1, phiAxp-2 and phiAxp-3.

2. An application of a bacteriophage for preventing and treating ginger stem base rot disease, comprising step 1, strain preparation; step 2, culture medium and reagent preparation; step 3, phage suspension preparation; step 4, activity determination and preservation; step 5, breeding and transplanting; step 6, phage inoculation; characterized in that: In the above step 1, the pathogenic bacteria Achromobacter xylosoxidans and bacteriophage are prepared; In the above step 2, the culture medium and related reagents required for preparing the bacterial solution are prepared; In the above step 3, a phage suspension is prepared and enriched; In the above step 4, the activity of the bacteriophage is measured and stored; In the above step 5, ginger is bred and transplanted; In the above step six, the phage inoculation is carried out by rhizome injection method.

3. The use of a bacteriophage for preventing and treating ginger stem base rot disease according to claim 2, characterized in that: In the step 1, Achromobacter xylosoxidans is isolated from the ginger stem tissue with serious disease, and a strong lytic phage is screened from its rhizosphere soil by a double-layer plate method, and one or more phages of phiAxp-1, phiAxp-2 and phiAxp-3 are prepared for joint culture, and the phage suspension is placed in a glycerol tube and stored in an ultra-low temperature freezer at -80°C.

4. The use of a bacteriophage for preventing and treating ginger stem base rot disease according to claim 2, characterized in that: In the step 2, NA liquid culture medium is prepared, the ingredients are 5 grams of peptone, 3 grams of beef extract, 10 grams of glucose and 800 milliliters of pure water, the pH is adjusted to 7.2-7.4, and autoclaved at 120°C for 45 minutes. NA solid culture medium, the ingredients are 5 grams of peptone, 3 grams of beef extract, 10 grams of glucose, 800 milliliters of pure water and 25 grams of agar, the pH is adjusted to 7.2-7.4, and autoclaved at 120°C for 45 minutes.

5. The use of a bacteriophage for preventing and treating ginger stem base rot disease according to claim 4, characterized in that: In the step 2, the selective culture medium is added in sequence to the NA liquid culture medium with 50 mg of crystal violet, 25 mg of bacitracin, 5 mg of penicillin, 50 mg of bacitracin and 100 mg of polybactin, and the buffer is added with 2 g of magnesium sulfate, 5 g of sodium chloride and 5 g of gelatin, 50 ml of the buffer base liquid, and pure water is added to make the volume to 1000 ml, and the mixture is sterilized under high pressure at 120° C. for 30 minutes.

6. The use of a bacteriophage for preventing and treating ginger stem base rot disease according to claim 3, characterized in that: In the step 3, the pathogenic bacteria Achromobacter xylosoxidans are inoculated and cultured on a selective culture medium, cultured in a constant temperature incubator at 32° C. for 36 hours, and high-quality colonies are selected and transferred to NA liquid culture medium, which is placed in a shaker device for shaking and culture for 48 hours.

7. The use of a bacteriophage for preventing and treating ginger stem base rot disease according to claim 6, characterized in that: In the step three, the phage suspension is taken out, 15 ml of the xylose-oxidizing Achromobacterium bacterial solution is taken and put into the NA solid culture medium to mix, and then poured on the culture medium plate, the phage suspension is connected to the plate, and cultured at 32° C. for 24 to 36 hours, during which the phage plaques are selected and transferred to the xylose-oxidizing Achromobacterium bacterial solution, shaken and allowed to stand for 15 minutes, and cultured at 32° C. for 12 to 15 hours to allow the phage to fully invade the xylose-oxidizing Achromobacterium for lysis and proliferation. The completely lysed culture solution contains a large amount of bacterial fragments in the form of limited filaments. The suspension is placed in a centrifuge at 12,000 revolutions per minute and centrifuged for 10 minutes, and the supernatant is collected and filtered through a filter membrane to obtain a phage suspension.

8. The use of a bacteriophage for preventing and treating ginger stem base rot disease according to claim 2, characterized in that: In the step 4, the quantity and activity of the phage suspension are determined, the concentration is diluted with a buffer solution, and freeze-dried bacterial powder is prepared and stored at ultra-low temperatures.

9. The use of a bacteriophage for preventing and treating ginger stem base rot disease according to claim 2, characterized in that: In the step 5, ginger seedlings are selected for breeding, and then the ginger seedlings are transplanted into a transplanting tray, and the soil matrix attached to the rhizomes is washed off during transplanting.

10. The use of a bacteriophage for preventing and treating ginger stem base rot disease according to claim 2, characterized in that: In the step six, the phage powder is prepared into a suspension by using a buffer solution, and then 75% alcohol is sprayed on the rhizome of ginger for disinfection using a spray gun, and the phage is injected and inoculated at the rhizome of ginger using a syringe. The inoculated ginger seedlings are covered with soil and cultivated for one week and then transferred to the greenhouse area. The phage suspension can be appropriately irrigated to the roots of the transplanting pit.

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