Ralstonia solanacearum and fermentation method for high-yield Ralstonia solanacearum bacteriophage

Abstract

The present invention discloses a fermentation method for Ralstonia solanacearum and a high-yield Ralstonia solanacearum phage, belonging to the field of microbial technology. The present invention obtains a new strain of Ralstonia solanacearum (Ralstonia solanacearum) BMB-Rs0001 by screening, and its preservation number is CCTCC NO: M20241876. This strain can significantly increase the explosiveness of Ralstonia solanacearum phage fermentation as a host bacteria. At the same time, the present invention improves the fermentation medium, fermentation temperature and feeding process, and ultimately greatly increases the fermentation yield of Ralstonia solanacearum phage, with the highest titer reaching 1.05×10 12 The Ralstonia solanacearum phage obtained by the method of the present invention significantly enhanced its storage stability at 4°C and 25°C over 180 days, and its control rate against bacterial wilt in plants increased from 72.82% to 90.51%.

Description

Technical Field

[0001] The present invention belongs to the technical field of microorganisms, and in particular relates to a fermentation method of Ralstonia solanacearum and a high-yield Ralstonia solanacearum bacteriophage. Background Art

[0002] With the extensive use of chemical pesticides, the problem of resistance of plant diseases and insect pests is becoming increasingly serious. Plant bacterial wilt (also known as "plant cancer") caused by Ralstonia solanacearum is widely distributed in countries around the world and can parasitize more than 200 genera of plants in more than 50 families, causing huge economic losses worldwide. The current methods for the prevention and control of bacterial wilt mainly include soil rotation, cultivation of disease-resistant plant varieties, chemical control, biological control, etc. These methods can reduce the occurrence of plant bacterial wilt to a certain extent, but the prevention and control effects are not ideal, resulting in difficulties in large-scale promotion. As a natural killer of bacteria, phage therapy is one of the most certain technical routes in the field of "reducing and replacing antibiotics". Because of its high host specificity and efficient plant pathogen lysis ability, the use of Ralstonia solanacearum phage pesticide products to prevent and control plant bacterial wilt has shown great advantages.

[0003] CN117946980A discloses a Ralstonia solanacearum phage RSP17, whose deposit number is CCTCCNO: M20232475. The fermentation titer of the phage is up to 2.4×10 10 CN117247908A discloses a Ralstonia solanacearum phage LPRS20, whose deposit number is CCTCC NO: M 20231194. The highest fermentation titer of the phage is 6.33×10 8 CN117187192A discloses a tobacco Ralstonia solanacearum phage PQ43, whose deposit number is CGMCC No 23813. The fermentation titer of the phage is up to 2.0×10 10 CN116144609A discloses a new type of bacteriophage for preventing and treating plant diseases caused by Ralstonia solanacearum and Pseudomonas syringae. Its deposit number is CCTCC M2020945. The highest fermentation titer of the bacteriophage is 9.1×10 10 PFU / mL.

[0004] Currently, research on Ralstonia solanacearum phage pesticides primarily focuses on resource development. Existing phages often suffer from low fermentation titers, long fermentation cycles, and significant fluctuations during fermentation, which, to a certain extent, contribute to high production costs. Furthermore, developing phage pesticides requires not only considering fermentation yield, which is a cost-limiting factor, but also comprehensively evaluating factors such as the phage's storage stability, which impacts the product's shelf life, and the biocontrol efficacy of the product. The storage stability of phages in a product is affected by multiple factors, including the solution's ionic strength and pH. Improving the phage's fermentation titer can lead to decreased storage stability due to changes in the culture method and culture medium. In addition, the effectiveness of using phages obtained after fermentation to control plant bacterial wilt is also related to the potency, activity and content of other metabolites (such as lytic enzymes) of the phage in the product. However, at the phage fermentation level, there is currently no literature that comprehensively evaluates and optimizes the relationship between the phage fermentation process and the fermentation potency and storage stability of the phage and the plant biocontrol effect. This further limits the industrialization and commercial promotion of the use of Ralstonia solanacearum phages to control plant bacterial wilt. Summary of the Invention

[0005] In response to the problems existing in the prior art, the purpose of the present invention is to provide a strain of Ralstonia solanacearum, and at the same time provide a fermentation method of Ralstonia solanacearum bacteriophage that is high-yielding, stable and has good disease prevention effect, thereby improving the fermentation titer and storage stability of Ralstonia solanacearum phage, and also enhancing the biocontrol effect of the high-yield phage on plant bacterial wilt.

[0006] In order to achieve the above technical objectives, the inventors screened host base bacteria for Ralstonia solanacearum phage fermentation to increase the burst of phages; screened fermentation medium with high yield of Ralstonia solanacearum phages to provide more suitable nutrition for the culture of host bacteria and phages, and changed the culture temperature at different fermentation stages, that is, during the Ralstonia solanacearum host culture period, the culture temperature was increased in stages to increase the biomass of the host bacteria for subsequent large-scale reproduction of phages and shorten the host bacteria culture time; during the phage culture period, the culture temperature was lowered in stages to be more suitable for the growth of phages. In addition, during the phage culture period, the problem of insufficient nutrition of the host bacteria at a certain stage of culture can be solved by adding nutrient culture medium in batches, further increasing the biomass of the host bacteria for phage reproduction. As the host bacteria are lysed to a certain stage, the addition of host bacteria in the logarithmic phase can provide a basis for phage reproduction again. In the later stage of phage culture, the release of phages can be increased by adding an inorganic salt mixture. The present invention improves the fermentation yield of Ralstonia solanacearum bacteriophage from multiple angles by screening host bacteria, varying temperature culture, and supplementing nutrient culture medium, host bacteria, and inorganic salt mixture. The storage stability and disease prevention effect of the Ralstonia solanacearum bacteriophage after the fermentation yield is improved are compared.

[0007] Specifically, the technical objectives of the present invention are achieved by developing a novel strain of Ralstonia solanacearum, named Ralstonia solanacearum BMB-Rs0001, which was deposited with the China Center for Type Culture Collection on September 2, 2024, with a deposit number of CCTCC NO: M20241876. This strain is used as a host for fermentation production of Ralstonia solanacearum phage, demonstrating high phage burst and fermentation yield.

[0008] It should be noted that the Ralstonia solanacearum BMB-Rs0001 strain of the present invention was obtained by screening for fermentative phage burst from over 800 strains housed at the National Key Laboratory of Agricultural Microbial Resources Discovery and Utilization at Huazhong Agricultural University. Its original source was the rhizosphere soil of diseased tomatoes in Zhuhai City, Guangdong Province. Furthermore, Ralstonia solanacearum BMB-Rs0001 has a small, rod-shaped cell morphology. Colonies grown on solid culture medium are pale yellow, with a smooth, opaque surface. It is aerobic, can reduce nitrate to nitrite, and ferment D-glucose to produce acid.

[0009] In addition, the present invention also provides a high-yield fermentation method for Ralstonia solanacearum phage, which can improve the fermentation titer and storage stability of Ralstonia solanacearum phage and enhance the biocontrol effect of the phage on plant bacterial wilt. The method comprises the following steps:

[0010] (1) Fermentation culture of the Ralstonia solanacearum host bacteria: a Ralstonia solanacearum BMB-Rs0001 seed solution was inoculated into a fermentation medium for host bacteria culture, and the dissolved oxygen (DO2) was controlled to be ≥40% by adjusting the stirring speed and the air intake. The fermentation temperature was controlled to be 30-33°C (preferably 32°C) from the 0th to the 4th hour, and the fermentation temperature was controlled to be 35-38°C (preferably 37°C) from the 4th to the 8th hour to obtain a host bacteria fermentation culture solution;

[0011] (2) Fermentation culture of Ralstonia solanacearum phage: inoculating the phage seed liquid into the host bacteria fermentation culture medium obtained in step (1) to carry out phage culture, controlling the dissolved oxygen (DO2) to 18-22% by adjusting the stirring speed and the air intake, the culture temperature to 28-35°C (preferably 32°C) from the 0th to the 4th hour, and the culture temperature to 25-30°C (preferably 28°C) from the 4th to the 8th hour; within the 2nd to the 4th hour, adding an equal amount of nutrient medium in 2-4 times (preferably 3 times), the total amount of nutrient medium added is 5-10% (preferably 8%) of the fermentation volume; within the 6th to the 8th hour, adding an inorganic salt mixture, the amount of the inorganic salt mixture added is 0.3-0.8% (preferably 0.5%) of the fermentation volume;

[0012] The components of the fermentation medium in step (1) include: fructose syrup, soybean meal, yeast extract, corn steep liquor powder, inositol, sodium glutamate, K2HPO4·3H2O, MgSO4·H2O;

[0013] The components of the nutrient medium in step (2) include: sucrose, sodium glutamate, inositol, and L-aspartic acid;

[0014] The components of the inorganic salt mixed solution in step (2) include: CaCl2·2H2O, K2HPO4·3H2O, KH2PO4, and MnCl2·4H2O.

[0015] The above-mentioned Ralstonia solanacearum BMB-Rs0001 seed solution can be prepared according to the following method: inoculating the active Ralstonia solanacearum BMB-Rs0001 bacterial solution into the host bacteria seed culture medium at a volume ratio of 0.5% to 1.5% (preferably 1%), controlling the culture temperature between 35°C and 38°C (preferably 37°C), setting the shaker speed to between 180rpm and 250rpm (preferably 220rpm), and culturing for 8h-24h (preferably 12h) to obtain the Ralstonia solanacearum BMB-Rs0001 seed solution.

[0016] The host bacteria seed culture medium comprises (g / L): 15-35 (preferably 20) sucrose, 4-6 (preferably 5) soy peptone, 0.2-0.4 (preferably 0.3) K2HPO4·3H2O, and 0.2-0.3 (preferably 0.25) MgSO4·H2O. The pH is adjusted to 6.5-7.5 (preferably 7.2), and the culture medium is sterilized at 0.11Mpa-0.12Mpa for 30 minutes to keep the culture medium in a sterile state.

[0017] In addition, the phage seed solution can be prepared as follows: inoculate the active Ralstonia solanacearum phage suspension at a volume ratio of 0.1% to 0.3% (preferably 0.2%) into the culture medium that has been cultured to the logarithmic phase (OD600 = 0.8-1.2, preferably 1.0) in a Ralstonia solanacearum BMB-Rs0001 seed liquid, control the culture temperature between 25°C and 32°C (preferably 28°C), set the shaker speed between 180rpm and 250rpm (preferably 220rpm), and culture for 4h-8h (preferably 6h) to obtain a phage seed liquid.

[0018] Further preferably, in the fermentation method for high-yield Ralstonia solanacearum phage, the concentration of the Ralstonia solanacearum BMB-Rs0001 seed solution in step (1) is OD 600 =0.8~1.2 (preferably OD 600 =1.0), and the inoculation amount is 5-10% (preferably 8%) in terms of volume percentage.

[0019] Further preferably, in the fermentation method for high-yield Ralstonia solanacearum phage as described above, the components of the fermentation medium in step (1) are: fructose syrup 30-80 g / L, soybean meal 20-40 g / L, yeast extract 3-8 g / L, corn steep liquor powder 8-12 g / L, inositol 1-2 g / L, sodium glutamate 0.5-1.5 g / L, K2HPO4·3H2O 0.8-1.2 g / L, MgSO4·H2O 0.2-0.6 g / L, and pH = 6.2-7.2. Still further preferably, the fermentation medium comprises the following components: fructose syrup 50 g / L, soybean meal 30 g / L, yeast extract 5 g / L, corn steep liquor powder 10 g / L, inositol 1.5 g / L, sodium glutamate 1 g / L, K2HPO4·3H2O 1.0 g / L, MgSO4·H2O 0.5 g / L, and pH = 6.5.

[0020] More preferably, in the fermentation method for high-yield Ralstonia solanacearum phage, the concentration of the phage seed solution in step (2) is 0.6 to 1.4×10 8 PFU / mL, the inoculum size is 0.5-2% (preferably 1%) by volume.

[0021] Further preferably, in the fermentation method for high-yield Ralstonia solanacearum phage, the components of the nutrient medium in step (2) are: sucrose 20-40 g / L, sodium glutamate 5-8 g / L, inositol 0.1-0.3 g / L, and L-aspartic acid 0.5-1.5 g / L. Still further preferably, the components of the nutrient medium are: sucrose 30 g / L, sodium glutamate 6 g / L, inositol 0.25 g / L, and L-aspartic acid 1 g / L.

[0022] Further preferably, in the fermentation method for high-yield Ralstonia solanacearum phage as described above, the components of the inorganic salt mixture in step (2) are: CaCl2·2H2O 0.5-1.5 g / L, K2HPO4·3H2O 0.25-0.6 g / L, KH2PO4 0.15-0.4 g / L, and MnCl2·4H2O 1.5-2.5 g / L. Still further preferably, the components of the inorganic salt mixture are: CaCl2·2H2O 1 g / L, K2HPO4·3H2O 0.5 g / L, KH2PO4 0.25 g / L, and MnCl2·4H2O 2 g / L.

[0023] Further preferably, in the fermentation method for high-yield Ralstonia solanacearum phage, in step (2), during the fermentation culture, when the viable cell value is ≤2.5 pF / cm, the host bacteria suspension is added. The host bacteria fermentation culture liquid can be the host bacteria fermentation culture liquid prepared in step (1).

[0024] Still more preferably, in the fermentation method for high-yield Ralstonia solanacearum phage, the concentration of the host bacterial suspension added in step (2) is 1.5≤OD 600 ≤2.5, the amount of host bacteria suspension added is 1-5% (preferably 3%) of the fermentation volume.

[0025] Further preferably, in the fermentation method for high-yield Ralstonia solanacearum phage as described above, the stirring speed during the cultivation process of step (1) and step (2) is in the range of 200 rpm to 800 rpm, and the air intake volume is in the range of 0.5 to 1.2 VVM.

[0026] Compared with the prior art, the fermentation method of Ralstonia solanacearum and Ralstonia solanacearum phage of the present invention has the following advantages and progress:

[0027] (1) The present invention obtained a new strain of Ralstonia solanacearum BMB-Rs0001 through screening. This strain can significantly increase the explosiveness of Ralstonia solanacearum phage fermentation as a host bacteria.

[0028] (2) The present invention obtains a fermentation medium with high yield of Ralstonia solanacearum phage through screening, which provides more suitable nutrition for the culture of host bacteria and phage.

[0029] (3) The present invention changes the culture temperature at different fermentation stages, that is, during the Ralstonia solanacearum host culture period, the culture temperature is increased in stages to increase the biomass of the host bacteria for subsequent large-scale reproduction of phages and shorten the host bacteria culture time; during the phage culture period, the culture temperature is lowered in stages to be more suitable for the growth of phages.

[0030] (4) The present invention first solves the problem of nutrient deficiency when the host bacteria grows to a certain stage by adding appropriate nutrient culture medium in batches, thereby increasing the biomass of the host bacteria for phage reproduction. Then, as the host bacteria lyse to a certain stage, the addition of host bacteria in the logarithmic phase can provide a basis for phage reproduction again. Finally, the addition of an inorganic salt mixture in the late stage of phage culture can increase the amount of phage particles released.

[0031] (5) The present invention significantly increases the fermentation yield of Ralstonia solanacearum phage, with the highest titer reaching 1.05×10 12 PFU / mL, and the total fermentation preparation time was only 16.8 hours.

[0032] (6) The present invention significantly improves the storage stability of Ralstonia solanacearum phage at 4°C and 25°C within 180 days.

[0033] (7) The present invention significantly improves the application effect of Ralstonia solanacearum phage in controlling plant bacterial wilt, and the control rate is increased from 72.82% to 90.51% compared with before technical optimization. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 : Flow chart of the experimental method for preparing high-yield, stable and efficient Ralstonia solanacearum phage of the present invention.

[0035] Figure 2 : Plaque image of bacteriophage vB_RsoP_BMB71.

[0036] Figure 3 : Colony map of Ralstonia solanacearum BMB-Rs0001 of the present invention.

[0037] Figure 4 : Photos of the biocontrol application effect of tomato bacterial wilt in the experimental group before optimization.

[0038] Figure 5 : Photos of the biocontrol application effect of tomato bacterial wilt in the optimized experimental group.

[0039] Figure 6 : Photos of indoor potted plant experiments to test the prevention and control of tomato bacterial wilt. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. In addition, the inventors selected a variety of Ralstonia solanacearum phages and host bacteria for research in the actual study of the fermentation method for improving the Ralstonia solanacearum phage, and only some of the results are presented below. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0041] In the following examples, R. solanacearum GMI1000 and FJAT-1458 were used as standard strains, and BMB-Rs3226 was isolated from the wild in Fujian Province. R. solanacearum phage Rs-sp22 was isolated from the rhizosphere soil of diseased tomatoes in Wenzhou, Zhejiang Province; Rs-sp56 was isolated from the rhizosphere soil of a tomato plant in Dalian, Liaoning Province; and Rs-sp90 was isolated from the rhizosphere soil of a pepper plant in Leping, Jiangxi Province. These R. solanacearum strains and their phages are all deposited at the National Key Laboratory of Agricultural Microbial Resources Discovery and Utilization, Huazhong Agricultural University.

[0042] Example 1: Host screening (constant temperature fermentation + different hosts + LB medium + no feed)

[0043] A fermentation method for Ralstonia solanacearum bacteriophage comprises the following steps:

[0044] (1) Host bacteria seed liquid culture: Different active Ralstonia solanacearum bacterial liquids were inoculated into the host bacteria seed liquid culture medium at a volume ratio of 1%, the culture temperature was controlled at 37°C, the shaking speed was set to 220 rpm, and the culture was carried out for 12 hours to obtain the host bacteria seed culture liquid.

[0045] The host bacteria seed culture medium comprises (g / L): 20g of sucrose, 5g of soy peptone, 0.3g of K2HPO4·3H2O, and 0.25g of MgSO4·H2O. The pH is adjusted to 7.2 and sterilized at 0.11-0.12 MPa for 30 minutes to keep the medium sterile.

[0046] (2) Phage seed culture: The active Ralstonia solanacearum phage vB_RsoP_BMB71 (deposit number: CCTCC NO: M 20231292) suspension was inoculated at a volume ratio of 0.2% until the culture reached the logarithmic phase (OD 600 =1.0), the culture temperature was controlled at 28°C, the shaker speed was set to 220 rpm, and the culture was carried out for 6 hours to obtain phage seed culture liquid.

[0047] (3) Fermentation culture of host bacteria: Different Ralstonia solanacearum seed liquids (OD 600 = 1.0) were inoculated into a fermenter filled with sterile LB medium at an inoculum volume ratio of 8% for host bacteria cultivation. The DO2 was controlled to be ≥ 40% by adjusting the stirring speed (200 rpm-800 rpm) and the air intake (0.5-1.2 VVM). The fermentation temperature was controlled at 37° C. during the host bacteria cultivation stage.

[0048] The LB culture medium includes (g / L): 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl. The pH is adjusted to 6.5 and sterilized at 0.11 MPa to 0.12 MPa for 30 minutes to keep the medium in a sterile state.

[0049] (4) Fermentation culture of phage: After culturing the above host bacteria for 8 hours, add phage seed liquid (about 10 8 The phage culture was continued by adjusting the stirring speed (200-800 rpm) and the air volume (0.5-1.2 VVM) to control the DO2 at around 20%. The culture temperature was controlled at 37°C during the phage culture stage.

[0050] (5) After the fermentation is completed, the Ralstonia solanacearum phage is isolated and extracted from the fermentation broth and the phage production (titer) is tested.

[0051] Table 1 shows the fermentation yields of the phages. The fermentation yields of the phage vB_RsoP_BMB71 were obtained under constant temperature and unfed-batch conditions using different Ralstonia solanacearum host bacteria and LB medium.

[0052] Table 1

[0053]

[0054] As can be seen from Table 1, by using different host bacteria to ferment phages at constant temperature under LB medium conditions, the host bacteria BMB-Rs0001 of the present invention can significantly increase the explosiveness of phage fermentation compared with other host bacteria.

[0055] Example 2: Screening of culture medium (constant temperature fermentation + host bacteria of the present invention + different culture media + no feed)

[0056] A fermentation method for Ralstonia solanacearum bacteriophage comprises the following steps:

[0057] (1) Host bacteria seed liquid culture: Active Ralstonia solanacearum BMB-Rs0001 bacterial liquid was inoculated into the host bacteria seed liquid culture medium at a volume ratio of 1%, the culture temperature was controlled at 37°C, the shaking speed was set to 220 rpm, and the culture was carried out for 12 h to obtain the host bacteria seed culture liquid.

[0058] The host bacteria seed culture medium comprises (g / L): 20g of sucrose, 5g of soy peptone, 0.3g of K2HPO4·3H2O, and 0.25g of MgSO4·H2O. The pH is adjusted to 7.2 and sterilized at 0.11-0.12 MPa for 30 minutes to keep the medium sterile.

[0059] (2) Phage seed liquid culture: The active Ralstonia solanacearum phage suspension was inoculated at a volume ratio of 0.2% until it reached the logarithmic phase (OD 600 =1.0) in a seed culture solution of Ralstonia solanacearum BMB-Rs0001, the culture temperature was controlled at 28° C., the shaking speed was set to 220 rpm, and the culture was carried out for 6 h to obtain a phage seed culture solution.

[0060] (3) Fermentation culture of host bacteria: The Ralstonia solanacearum seed solution (OD 600 = 1.0) was inoculated at an inoculum volume ratio of 8% into a fermenter containing sterile fermentation medium (and LB medium) for host bacteria cultivation. The DO2 was controlled to be ≥ 40% by adjusting the stirring speed (200 rpm-800 rpm) and the air intake (0.5-1.2 VVM). The fermentation temperature was controlled at 37° C. during the host bacteria cultivation stage.

[0061] The fermentation medium comprises (g / L): 50g of fructose syrup, 30g of soybean meal, 5g of yeast extract, 10g of corn steep liquor powder, 1.5g of inositol, 1g of sodium glutamate, 1.0g of K2HPO4·3H2O, and 0.5g of MgSO4·H2O. The pH value is adjusted to 6.5, and the medium is sterilized at 0.11Mpa-0.12Mpa for 30 minutes to keep the medium in a sterile state.

[0062] The LB culture medium includes (g / L): 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl. The pH is adjusted to 6.5 and sterilized at 0.11 MPa to 0.12 MPa for 30 minutes to keep the medium in a sterile state.

[0063] (4) Fermentation culture of phage: After the host bacteria are cultured for 8 hours, the phage seed solution (about 10 8 The phage culture was continued by adjusting the stirring speed (200-800 rpm) and the air volume (0.5-1.2 VVM) to control the DO2 at around 20%. The culture temperature was controlled at 37°C during the phage culture stage.

[0064] (5) After the fermentation is completed, the Ralstonia solanacearum phage is isolated and extracted from the fermentation broth and the phage production (titer) is tested.

[0065] The fermentation yields of the phage are shown in Table 2 below, which shows the fermentation yields of the phage vB_RsoP_BMB71 under constant temperature and unfed fermentation conditions under different fermentation medium conditions:

[0066] Table 2

[0067]

[0068] As can be seen from Table 2, when phages are fermented at a constant temperature under the host bacteria conditions of the present invention using different culture media, the fermentation medium of the present invention can significantly increase the fermentation yield of phages compared to LB medium.

[0069] Example 3: Variable temperature fermentation (variable temperature fermentation + host of the present invention + culture medium of the present invention + no feed)

[0070] A fermentation method for Ralstonia solanacearum bacteriophage comprises the following steps:

[0071] (1) Host bacteria seed liquid culture: Active Ralstonia solanacearum BMB-Rs0001 bacterial liquid was inoculated into the host bacteria seed liquid culture medium at a volume ratio of 1%, the culture temperature was controlled at 37°C, the shaking speed was set to 220 rpm, and the culture was carried out for 12 h to obtain the host bacteria seed culture liquid.

[0072] The host bacteria seed culture medium comprises (g / L): 20g of sucrose, 5g of soy peptone, 0.3g of K2HPO4·3H2O, and 0.25g of MgSO4·H2O. The pH value is adjusted to 7.2, and the culture medium is sterilized at 0.11-0.12 MPa for 30 minutes to keep the culture medium in a sterile state.

[0073] (2) Phage seed liquid culture: The active Ralstonia solanacearum phage suspension was inoculated at a volume ratio of 0.2% until it reached the logarithmic phase (OD 600 =1.0), the culture temperature was controlled at 28°C, the shaking speed was set to 220 rpm, and the culture was carried out for 6 hours to obtain the phage seed culture solution.

[0074] (3) Fermentation culture of host bacteria: The Ralstonia solanacearum seed solution (OD 600= 1.0) was inoculated into a fermenter containing sterile fermentation medium at an inoculum volume ratio of 8% for host bacteria cultivation. The DO2 was controlled to be ≥ 40% by adjusting the stirring speed (200 rpm-800 rpm) and the air intake volume (0.5-1.2 VVM). The fermentation temperature was controlled at 32° C. for 0-4 hours and at 37° C. for 4-8 hours during the host bacteria cultivation stage.

[0075] The fermentation medium comprises (g / L): 50g of fructose syrup, 30g of soybean meal, 5g of yeast extract, 10g of corn steep liquor powder, 1.5g of inositol, 1g of sodium glutamate, 1.0g of K2HPO4·3H2O, and 0.5g of MgSO4·H2O. The pH value is adjusted to 6.5, and the medium is sterilized at 0.11Mpa-0.12Mpa for 30 minutes to keep the medium in a sterile state.

[0076] (4) Fermentation culture of phage: After the host bacteria are cultured for 8 hours, the phage seed solution (about 10 8 PFU / mL) was inoculated at a 1% inoculum size into a fermenter containing host bacterial culture medium, and phage culture was continued (recorded as the 0th hour of phage culture). The stirring speed (200 rpm-800 rpm) and the air intake volume (0.5-1.2 VVM) were adjusted to control DO2 at around 20%. The culture temperature for the 0th to 4th hour of phage culture was 32°C, and the culture temperature for the 4th to 8th hour of phage culture was 28°C.

[0077] (5) After the fermentation is completed, the Ralstonia solanacearum phage is isolated and extracted from the fermentation broth and the phage production (titer) is tested.

[0078] Table 3 below shows the fermentation yield of the phage vB_RsoP_BMB71, which was obtained by performing variable temperature and unfed-batch fermentation using Ralstonia solanacearum as the host bacteria and the fermentation medium conditions of the present invention.

[0079] Table 3

[0080]

[0081] As can be seen from Table 3, the use of variable temperature culture of phage under the conditions of the host bacteria and culture medium of the present invention can significantly increase the fermentation yield of phage compared to constant temperature (37°C) culture, and at the same time shorten the fermentation time.

[0082] Example 4: Supplementation of nutrient medium (variable temperature fermentation + medium of the present invention + host of the present invention + supplementation of nutrient medium)

[0083] A fermentation method for Ralstonia solanacearum bacteriophage comprises the following steps:

[0084] (1) Host bacteria seed liquid culture: Active Ralstonia solanacearum BMB-Rs0001 bacterial liquid was inoculated into the host bacteria seed liquid culture medium at a volume ratio of 1%, the culture temperature was controlled at 37°C, the shaking speed was set to 220 rpm, and the culture was carried out for 12 h to obtain the host bacteria seed culture liquid.

[0085] The host bacteria seed culture medium comprises (g / L): 20g of sucrose, 5g of soy peptone, 0.3g of K2HPO4·3H2O, and 0.25g of MgSO4·H2O. The pH value is adjusted to 7.2, and the culture medium is sterilized at 0.11-0.12 MPa for 30 minutes to keep the culture medium in a sterile state.

[0086] (2) Phage seed liquid culture: The active Ralstonia solanacearum phage suspension was inoculated at a volume ratio of 0.2% until it reached the logarithmic phase (OD 600 =1.0) in a Ralstonia solanacearum seed culture solution and the culture temperature was controlled at 28°C, the shaking speed was set to 220 rpm, and the culture was carried out for 6 hours to obtain a phage seed culture solution.

[0087] (3) Fermentation culture of host bacteria: The Ralstonia solanacearum seed solution (OD 600 = 1.0) was inoculated into a fermenter containing sterile fermentation medium at an inoculum volume ratio of 8% for host bacteria cultivation. The DO2 was controlled to be ≥ 40% by adjusting the stirring speed (200 rpm-800 rpm) and the air intake volume (0.5-1.2 VVM). The fermentation temperature was controlled at 32° C. for 0-4 hours and at 37° C. for 4-8 hours during the host bacteria cultivation stage.

[0088] The fermentation medium comprises (g / L): 50g of fructose syrup, 30g of soybean meal, 5g of yeast extract, 10g of corn steep liquor powder, 1.5g of inositol, 1g of sodium glutamate, 1.0g of K2HPO4·3H2O, and 0.5g of MgSO4·H2O. The pH value is adjusted to 6.5, and the medium is sterilized at 0.11Mpa-0.12Mpa for 30 minutes to keep the medium in a sterile state.

[0089] (4) Fermentation culture of phage: After the host bacteria are cultured for 8 hours, the phage seed solution (about 10 8 PFU / mL) was inoculated at a 1% inoculum size into a fermenter containing host bacterial culture medium, and phage culture was continued (recorded as the 0th hour of phage culture). The stirring speed (200 rpm-800 rpm) and the air intake volume (0.5-1.2 VVM) were adjusted to control DO2 at around 20%. The culture temperature for the 0th to 4th hour of phage culture was 32°C, and the culture temperature for the 4th to 8th hour of phage culture was 28°C.

[0090] (5) Fed-batch fermentation of phage: During the 2nd to 4th hour of phage culture, equal amounts of nutrient medium were added in three batches to increase the growth of the host bacteria. The total amount of nutrient medium added was 8% of the fermentation volume.

[0091] The components of the nutrient medium are (g / L): sucrose 30, sodium glutamate 6, inositol 0.25, L-aspartic acid 1.

[0092] (6) After the fermentation is completed, the Ralstonia solanacearum phage is isolated and extracted from the fermentation broth and the phage production (titer) is tested.

[0093] Table 4 below shows the fermentation yield of phage vB_RsoP_BMB71 using Ralstonia solanacearum as the host bacteria and the fermentation medium conditions of the present invention, with varying temperatures and supplemented nutrient medium:

[0094] Table 4

[0095]

[0096]

[0097] As can be seen from Table 4, under the host bacteria, culture medium and temperature-variable culture conditions of the present invention, the addition of nutrient medium can significantly increase the fermentation yield of phage compared to no feeding.

[0098] Example 5: Supplementation of host bacteria (variable temperature fermentation + culture medium of the present invention + host of the present invention + supplementation of host bacteria)

[0099] A fermentation method for Ralstonia solanacearum bacteriophage comprises the following steps:

[0100] (1) Host bacteria seed liquid culture: Active Ralstonia solanacearum BMB-Rs0001 bacterial liquid was inoculated into the host bacteria seed liquid culture medium at a volume ratio of 1%, the culture temperature was controlled at 37°C, the shaking speed was set to 220 rpm, and the culture was carried out for 12 h to obtain the host bacteria seed culture liquid.

[0101] The host bacteria seed culture medium comprises (g / L): 20g of sucrose, 5g of soy peptone, 0.3g of K2HPO4·3H2O, and 0.25g of MgSO4·H2O. The pH value is adjusted to 7.2, and the culture medium is sterilized at 0.11-0.12 MPa for 30 minutes to keep the culture medium in a sterile state.

[0102] (2) Phage seed liquid culture: The active Ralstonia solanacearum phage suspension was inoculated at a volume ratio of 0.2% until it reached the logarithmic phase (OD 600=1.0), the culture temperature was controlled at 28°C, the shaking speed was set to 220 rpm, and the culture was carried out for 6 hours to obtain the phage seed culture solution.

[0103] (3) Fermentation culture of host bacteria: The Ralstonia solanacearum seed solution (OD 600 = 1.0) was inoculated into a fermenter containing sterile fermentation medium at an inoculum volume ratio of 8% for host bacteria cultivation. The DO2 was controlled to be ≥ 40% by adjusting the stirring speed (200 rpm-800 rpm) and the air intake volume (0.5-1.2 VVM). The fermentation temperature was controlled at 32° C. for 0-4 hours and at 37° C. for 4-8 hours during the host bacteria cultivation stage.

[0104] The fermentation medium comprises (g / L): 50g of fructose syrup, 30g of soybean meal, 5g of yeast extract, 10g of corn steep liquor powder, 1.5g of inositol, 1g of sodium glutamate, 1.0g of K2HPO4·3H2O, and 0.5g of MgSO4·H2O. The pH value is adjusted to 6.5, and the medium is sterilized at 0.11Mpa-0.12Mpa for 30 minutes to keep the medium in a sterile state.

[0105] (4) Fermentation culture of phage: After the host bacteria are cultured for 8 hours, the phage seed solution (about 10 8 PFU / mL) was inoculated into a fermenter containing the host bacteria culture medium at an inoculum size of 1%, and phage culture was continued (recorded as the 0th hour of phage culture). The stirring speed (200 rpm-800 rpm) and the air intake volume (0.5-1.2 VVM) were adjusted to control DO2 at around 20%. The culture temperature for the 0th to 4th hour of phage culture was 32°C, and the culture temperature for the 4th to 8th hour of phage culture was 28°C.

[0106] (5) Phage fed-batch fermentation: During the phage culture stage, a certain bacterial concentration (1.5≤OD 600 ≤2.5) of host bacteria suspension until it is completed, and the total added volume of the host bacteria suspension accounts for 3% of the fermentation volume.

[0107] (6) After the fermentation is completed, the Ralstonia solanacearum phage is isolated and extracted from the fermentation broth and the phage production (titer) is tested.

[0108] Table 5 below shows the fermentation yield of phage vB_RsoP_BMB71 using Ralstonia solanacearum as the host bacteria and the fermentation medium conditions of the present invention, with temperature changes and supplementation of host bacteria culture fluid:

[0109] Table 5

[0110]

[0111] As can be seen from Table 5, the addition of host bacteria under the host bacteria, culture medium and temperature-variable culture conditions of the present invention can significantly increase the fermentation yield of phage compared to no feeding.

[0112] Example 6: Addition of inorganic salts (variable temperature fermentation + culture medium of the present invention + host of the present invention + addition of inorganic salts)

[0113] A fermentation method for Ralstonia solanacearum bacteriophage comprises the following steps:

[0114] (1) Host bacteria seed liquid culture: Active Ralstonia solanacearum BMB-Rs0001 bacterial liquid was inoculated into the host bacteria seed liquid culture medium at a volume ratio of 1%, the culture temperature was controlled at 37°C, the shaking speed was set to 220 rpm, and the culture was carried out for 12 h to obtain the host bacteria seed culture liquid.

[0115] The host bacteria seed culture medium comprises (g / L): 20g of sucrose, 5g of soy peptone, 0.3g of K2HPO4·3H2O, and 0.25g of MgSO4·H2O. The pH value is adjusted to 7.2, and the culture medium is sterilized at 0.11-0.12 MPa for 30 minutes to keep the culture medium in a sterile state.

[0116] (2) Phage seed liquid culture: The active Ralstonia solanacearum phage suspension was inoculated at a volume ratio of 0.2% until it reached the logarithmic phase (OD 600 =1.0), the culture temperature was controlled at 28°C, the shaking speed was set to 220 rpm, and the culture was carried out for 6 hours to obtain the phage seed culture solution.

[0117] (3) Fermentation culture of host bacteria: The Ralstonia solanacearum seed solution (OD 600 = 1.0) was inoculated into a fermenter containing sterile fermentation medium at an inoculum volume ratio of 8% for host bacteria cultivation. The DO2 was controlled to be ≥ 40% by adjusting the stirring speed (200 rpm-800 rpm) and the air intake volume (0.5-1.2 VVM). The fermentation temperature was controlled at 32° C. for 0-4 hours and at 37° C. for 4-8 hours during the host bacteria cultivation stage.

[0118] The fermentation medium comprises (g / L): 50g of fructose syrup, 30g of soybean meal, 5g of yeast extract, 10g of corn steep liquor powder, 1.5g of inositol, 1g of sodium glutamate, 1.0g of K2HPO4·3H2O, and 0.5g of MgSO4·H2O. The pH value is adjusted to 6.5, and the medium is sterilized at 0.11Mpa-0.12Mpa for 30 minutes to keep the medium in a sterile state.

[0119] (4) Fermentation culture of phage: After the host bacteria are cultured for 8 hours, the phage seed solution (about 10 8PFU / mL) was inoculated at a 1% inoculum size into a fermenter containing host bacterial culture medium, and phage culture was continued (recorded as the 0th hour of phage culture). The stirring speed (200 rpm-800 rpm) and the air intake volume (0.5-1.2 VVM) were adjusted to control DO2 at around 20%. The culture temperature for the 0th to 4th hour of phage culture was 32°C, and the culture temperature for the 4th to 8th hour of phage culture was 28°C.

[0120] (5) Phage fed-batch fermentation: 2-4 hours before the end of phage culture (i.e., 6-8 hours after phage culture), add an inorganic salt mixture, and the amount of the inorganic salt mixture added is 0.5% of the total fermentation volume.

[0121] The components of the inorganic salt mixture are (g / L): CaCl2·2H2O 1, K2HPO4·3H2O 0.5, KH2PO4 0.25, MnCl2·4H2O2.

[0122] (6) After the fermentation is completed, the Ralstonia solanacearum phage is isolated and extracted from the fermentation broth and the phage production (titer) is tested.

[0123] Table 6 below shows the fermentation yield of bacteriophage vB_RsoP_BMB71 using Ralstonia solanacearum as the host bacteria and the fermentation medium under the conditions of the present invention, with temperature change and addition of an inorganic salt mixture:

[0124] Table 6

[0125]

[0126] As can be seen from Table 6, the addition of the inorganic salt mixture under the host bacteria and culture medium and temperature-variable culture conditions of the present invention can significantly increase the fermentation yield of phage compared to no feeding.

[0127] Example 7: Combination of the present invention (variable temperature fermentation + host of the present invention + supplementation of nutrient solution / host bacteria / inorganic salts)

[0128] A fermentation method for Ralstonia solanacearum bacteriophage comprises the following steps:

[0129] (1) Host bacteria seed liquid culture: Active Ralstonia solanacearum BMB-Rs0001 bacterial liquid was inoculated into the host bacteria seed liquid culture medium at a volume ratio of 1%, the culture temperature was controlled at 37°C, the shaking speed was set to 220 rpm, and the culture was carried out for 12 h to obtain the host bacteria seed culture liquid.

[0130] The host bacteria seed culture medium comprises (g / L): 20g of sucrose, 5g of soy peptone, 0.3g of K2HPO4·3H2O, and 0.25g of MgSO4·H2O. The pH value is adjusted to 7.2, and the culture medium is sterilized at 0.11-0.12 MPa for 30 minutes to keep the culture medium in a sterile state.

[0131] (2) Phage seed liquid culture: The active Ralstonia solanacearum phage suspension was inoculated at a volume ratio of 0.2% until it reached the logarithmic phase (OD 600 =1.0), the culture temperature was controlled at 28°C, the shaking speed was set to 220 rpm, and the culture was carried out for 6 hours to obtain the phage seed culture solution.

[0132] (3) Fermentation culture of host bacteria: The Ralstonia solanacearum seed solution (OD 600 = 1.0) was inoculated into a fermenter containing sterile fermentation medium at an inoculum volume ratio of 8% for host bacteria cultivation. The DO2 was controlled to be ≥ 40% by adjusting the stirring speed (200 rpm-800 rpm) and the air intake volume (0.5-1.2 VVM). The fermentation temperature was controlled at 32° C. for 0-4 hours and at 37° C. for 4-8 hours during the host bacteria cultivation stage.

[0133] The fermentation medium comprises (g / L): 50g of fructose syrup, 30g of soybean meal, 5g of yeast extract, 10g of corn steep liquor powder, 1.5g of inositol, 1g of sodium glutamate, 1.0g of K2HPO4·3H2O, and 0.5g of MgSO4·H2O. The pH value is adjusted to 6.5, and the medium is sterilized at 0.11Mpa-0.12Mpa for 30 minutes to keep the medium in a sterile state.

[0134] (4) Fermentation culture of phage: After the host bacteria are cultured for 8 hours, the phage seed solution (about 10 8 PFU / mL) was inoculated at a 1% inoculum size into a fermenter containing host bacterial culture medium, and phage culture was continued (recorded as the 0th hour of phage culture). The stirring speed (200 rpm-800 rpm) and the air intake volume (0.5-1.2 VVM) were adjusted to control DO2 at around 20%. The culture temperature for the 0th to 4th hour of phage culture was 32°C, and the culture temperature for the 4th to 8th hour of phage culture was 28°C.

[0135] (5) Fed-batch fermentation of phage: During the 2nd to 4th hour of phage culture, add equal amounts of nutrient medium in 3 batches to increase the growth of the host bacteria. The total amount of nutrient medium added is 8% of the fermentation volume. During the phage culture stage, a certain bacterial concentration (1.5≤OD 600≤2.5) of host bacterial suspension, until it is complete, the host bacterial suspension is 3% of the total fermentation volume. 2-4 hours before the end of phage culture (i.e., 6-8 hours of phage culture), add inorganic salt mixture, the amount of inorganic salt mixture added is 0.5% of the total fermentation volume.

[0136] The components of the nutrient medium are (g / L): sucrose 30, sodium glutamate 6, inositol 0.25, L-aspartic acid 1.

[0137] The components of the inorganic salt mixture are (g / L): CaCl2·2H2O 1, K2HPO4·3H2O 0.5, KH2PO4 0.25, MnCl2·4H2O2.

[0138] (6) After the fermentation is completed, the Ralstonia solanacearum phage is isolated and extracted from the fermentation broth and the phage production (titer) is tested.

[0139] Table 7 below shows the fermentation yield of the bacteriophage vB_RsoP_BMB71, which was obtained by performing variable temperature fermentation and supplementing the nutrient medium, host bacteria, and inorganic salt mixture under the conditions of the present invention using Ralstonia solanacearum as the host bacteria and fermentation medium.

[0140] Table 7

[0141]

[0142] As can be seen from Table 7 above, the addition of nutrient medium, host bacteria and inorganic salt mixture has a synergistic effect under the host bacteria and culture medium and temperature-variable culture conditions of the present invention. Compared with no feeding, the method of the present invention can significantly increase the fermentation yield of phage.

[0143] Example 8: Other phage + combination of the present invention / control scheme

[0144] A fermentation method for Ralstonia solanacearum bacteriophage comprises the following steps:

[0145] (1) Host bacteria seed liquid culture: Different active Ralstonia solanacearum bacterial liquids were inoculated into the host bacteria seed liquid culture medium at a volume ratio of 1%, the culture temperature was controlled at 37°C, the shaking speed was set to 220 rpm, and the culture was carried out for 12 hours to obtain the host bacteria seed culture liquid.

[0146] The host bacteria seed culture medium comprises (g / L): 20g of sucrose, 5g of soy peptone, 0.3g of K2HPO4·3H2O, and 0.25g of MgSO4·H2O. The pH value is adjusted to 7.2, and the culture medium is sterilized at 0.11-0.12 MPa for 30 minutes to keep the culture medium in a sterile state.

[0147] (2) Phage seed liquid culture: Different active Ralstonia solanacearum phage suspensions were inoculated at a volume ratio of 0.2% to the culture medium until the logarithmic phase (OD 600 =1.0), the culture temperature was controlled at 28°C, the shaker speed was set to 220 rpm, and the culture was carried out for 6 hours to obtain phage seed culture liquid.

[0148] (3) Fermentation culture of host bacteria: Different Ralstonia solanacearum seed liquids (OD 600 = 1.0) were inoculated at an inoculum volume ratio of 8% into a fermentor tank filled with sterile fermentation medium (or LB medium) for host bacteria cultivation. The DO2 was controlled to be ≥ 40% by adjusting the stirring speed (200 rpm-800 rpm) and the air intake volume (0.5-1.2 VVM). The fermentation temperature of the host bacteria cultivation stage was controlled at 32° C. for 0-4 hours and 37° C. for 4-8 hours (or fermentation was performed at a constant temperature of 37° C.).

[0149] The fermentation medium comprises (g / L): 50g of fructose syrup, 30g of soybean meal, 5g of yeast extract, 10g of corn steep liquor powder, 1.5g of inositol, 1g of sodium glutamate, 1.0g of K2HPO4·3H2O, and 0.5g of MgSO4·H2O. The pH value is adjusted to 6.5, and the medium is sterilized at 0.11Mpa-0.12Mpa for 30 minutes to keep the medium in a sterile state.

[0150] The LB culture medium includes (g / L): 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl. The pH is adjusted to 6.5 and sterilized at 0.11 MPa to 0.12 MPa for 30 minutes to keep the medium in a sterile state.

[0151] (4) Fermentation culture of phage: After the host bacteria are cultured for 8 hours, different phage seed solutions (about 10 8 PFU / mL) were inoculated at a 1% inoculation rate into a fermenter containing host bacterial culture medium, and phage culture was continued (recorded as the 0th hour of phage culture). The stirring speed (200 rpm-800 rpm) and the air intake volume (0.5-1.2 VVM) were adjusted to control DO2 at around 20%. The temperature was controlled at 32°C for the 0th to 4th hour of phage culture and at 28°C for the 4th to 8th hour of phage culture (or a constant temperature of 37°C for fermentation).

[0152] (5) Fed-batch fermentation of phage: During the 2nd to 4th hour of phage culture, an equal amount of nutrient medium was added in three batches to increase the growth of the host bacteria. The total amount of nutrient medium added was 8% of the fermentation volume. During the phage culture stage, a certain bacterial concentration (1.5≤OD 600≤2.5) of host bacterial suspension until it is replenished, and the amount of host bacterial suspension added is 3% of the total fermentation volume. 2-4 hours before the end of phage culture (i.e., 6-8 hours of phage culture), add inorganic salt mixture, and the amount of inorganic salt mixture added is 0.5% of the total fermentation volume.

[0153] The components of the nutrient medium are (g / L): sucrose 30, sodium glutamate 6, inositol 0.25, L-aspartic acid 1.

[0154] The components of the inorganic salt mixture are (g / L): CaCl2·2H2O 1, K2HPO4·3H2O 0.5, KH2PO4 0.25, MnCl2·4H2O2.

[0155] (7) After the fermentation is completed, the Ralstonia solanacearum phage is isolated and extracted from the fermentation broth and the phage production (titer) is tested.

[0156] The fermentation yields of different phages are shown in Table 8 below, which shows the fermentation yields of different phages under the conditions of the present invention, Ralstonia solanacearum, and fermentation medium, with varying temperatures and supplementation of nutrient medium, host bacteria, and inorganic salt mixture:

[0157] Table 8

[0158]

[0159] As can be seen from Table 8 above, the host bacteria and fermentation method of the present invention also have the effect of significantly improving the fermentation yield in the fermentation application of the other three phages.

[0160] Example 9: Phage Stability Study

[0161] The phage sample No. 1 obtained by fermentation in Example 7 (1.05×10 12 PFU / mL) was used as the optimized experimental group, and the phage sample No. 2 obtained by fermentation in Example 1 (6.91×10 8 PFU / mL) was used as the experimental group before optimization. The above samples were aliquoted into 2 mL centrifuge tubes and then placed in a 4°C refrigerator and a 25°C incubator. One sample was taken out every 30 days for phage titer testing. The test results within 180 days are shown in Tables 9 and 10 below (unit: PFU / mL).

[0162] Table 9

[0163]

[0164] Table 10

[0165]

[0166] As can be seen from Tables 9 and 10 above, the optimized phage sample lost 0.13 log in potency after storage at 4°C for 180 days. 10 (0.13 orders of magnitude), and the potency loss after storage at 25°C for 180 days was 1.21 log 10 (1.21 orders of magnitude); the phage before optimization lost 1.1 log in potency after storage at 4°C for 180 days. 10 (1.1 orders of magnitude), and the potency loss was 4.08 log after storage at 25°C for 180 days. 10 (4.08 orders of magnitude), it is obvious that the loss rate of the optimized phage is significantly lower than that before optimization, which proves that the Ralstonia solanacearum phage optimized by the present invention has better storage stability.

[0167] Example 10: Study on the biocontrol effect of bacteriophage

[0168] The phage sample No. 1 obtained by fermentation in Example 7 (1.05×10 12 PFU / mL) was used as the optimized experimental group, and the phage sample No. 2 obtained by fermentation in Example 1 (6.91×10 8 PFU / mL) as the experimental group before optimization, and the above samples were diluted to the same concentration (1.0×10 8 PFU / mL) and was used in indoor pot experiments to test the effectiveness of Ralstonia solanacearum phages in preventing and controlling tomato bacterial wilt before and after fermentation optimization.

[0169] The experiment was set up with 4 groups: (1) clear water control; (2) negative control inoculated with only the pathogenic bacterium BMB-Rs3226 of Ralstonia solanacearum; (3) inoculated with the pathogenic bacterium BMB-Rs3226 of Ralstonia solanacearum and used the optimized prophage (prepared by diluting the sample No. 2 in Example 1) for prevention and treatment; (4) inoculated with the pathogenic bacterium BMB-Rs3226 of Ralstonia solanacearum and used the optimized postphage (prepared by diluting the sample No. 1 in Example 7) for prevention and treatment. The experiment selected tomato seedlings with three leaves and one heart that had relatively consistent growth and transplanted them into pots for the experiment. Each group had 10 plants and 4 groups were tested in parallel. The OD 600 The tomato plants were inoculated with 0.8-1.2% pathogens by root irrigation method, and the inoculation volume of pathogens was 40mL / pot. 8 PFU / mL) of phage were used for biological control. The roots of tomatoes were irrigated with phage three times on days 0, 7, and 14 after transplanting. The dosage of phage was 40 mL / pot each time. The plants were cultured at 30°C with alternating light and dark conditions of 12 h for 28 days. The incidence of bacterial wilt of tomatoes was observed (see Figure 4-Figure 6 ), the incidence and control rates of tomato bacterial wilt are shown in Table 11 below.

[0170] Table 11

[0171]

[0172] As can be seen from Table 11 above, under the premise of being diluted to the same concentration, the optimized phage sample has a better effect on the prevention and control of tomato bacterial wilt than the sample before optimization, and the 28-day control rate is increased from 72.73% to 90.48%, indicating that the Ralstonia solanacearum phage obtained by the method of the present invention has higher plant disease prevention activity.

Claims

1. A fermentation method for high-yield Ralstonia solanacearum phage, characterized in that: The method comprises the following steps: (1) Fermentation culture of Ralstonia solanacearum host bacteria: Ralstonia solanacearum with the preservation number of CCTCC NO: M20241876 ( Ralstonia solanacearum ) BMB-Rs0001 seed liquid was inoculated into a fermentation medium for host bacterial culture, and the dissolved oxygen was controlled to be ≥40% by adjusting the stirring speed and air intake volume. The fermentation temperature was controlled at 30-33° C. from hour 0 to hour 4, and at 35-38° C. from hour 4 to hour 8, to obtain a host bacterial fermentation culture liquid; (2) Fermentation culture of Ralstonia solanacearum phage: inoculating the phage seed liquid into the host bacteria fermentation culture medium obtained in step (1) for phage culture, controlling the dissolved oxygen to 18-22% by adjusting the stirring speed and the air intake, the culture temperature to 28-35°C from hour 0 to hour 4, and the culture temperature to 25-30°C from hour 4 to hour 8; within the second to fourth hour, adding an equal amount of nutrient culture medium in 2-4 times, the total amount of nutrient culture medium added is 5-10% of the fermentation volume; within the sixth to eighth hour, adding an inorganic salt mixture, the amount of the inorganic salt mixture added is 0.3-0.8% of the fermentation volume; The components of the fermentation medium in step (1) include: fructose syrup, soybean meal, yeast extract, corn steep liquor powder, inositol, sodium glutamate, K2HPO4•3H2O, MgSO4•H2O; The components of the nutrient medium in step (2) include: sucrose, sodium glutamate, inositol, and L-aspartic acid; The components of the inorganic salt mixture in step (2) include: CaCl2•2H2O, K2HPO4•3H2O, KH2PO4, MnCl2•4H2O; During the fermentation culture in step (2), when the viable cell count is ≤2.5 pF / cm, the host bacterial suspension is added.

2. The fermentation method for high-yield Ralstonia solanacearum phage according to claim 1, characterized in that: The Ralstonia solanacearum ( Ralstonia solanacearum ) The concentration of BMB-Rs0001 seed solution is OD 600 =0.8~1.2, and the inoculation amount is 5~10%.

3. The fermentation method for high-yield Ralstonia solanacearum phage according to claim 1, characterized in that: The components of the fermentation medium in step (1) are as follows: fructose syrup 30-80 g / L, soybean meal 20-40 g / L, yeast extract 3-8 g / L, corn steep liquor powder 8-12 g / L, inositol 2 g / L, sodium glutamate 0.5-1.5 g / L, K2HPO4•3H2O 0.8-1.2 g / L, MgSO4•H2O 0.2-0.6 g / L, pH=6.2-7.

2.

4. The fermentation method for high-yield Ralstonia solanacearum phage according to claim 1, characterized in that: The concentration of the phage seed solution in step (2) is 0.6~1.4×10 8 PFU / mL, and the inoculum size was 0.5~2%.

5. The fermentation method for high-yield Ralstonia solanacearum phage according to claim 1, characterized in that: The components of the nutrient medium in step (2) are as follows: sucrose 20-40 g / L, sodium glutamate 5-8 g / L, inositol 0.1-0.3 g / L, and L-aspartic acid 0.5-1.5 g / L.

6. The fermentation method for high-yield Ralstonia solanacearum phage according to claim 1, characterized in that: The component contents of the inorganic salt mixture in step (2) are: CaCl2•2H2O 0.5~1.5g / L, K2HPO4•3H2O 0.25~0.6g / L, KH2PO4 0.15~0.4g / L, and MnCl2•4H2O 1.5~2.5g / L.

7. The fermentation method for high-yield Ralstonia solanacearum phage according to claim 1, characterized in that: The concentration of the host bacterial suspension added in step (2) is 1.5≤OD 600 ≤2.5, the amount of host bacteria suspension added is 1~5% of the fermentation volume.

8. The fermentation method for high-yield Ralstonia solanacearum phage according to claim 1, characterized in that: The stirring speed during the cultivation process of step (1) and step (2) is in the range of 200 rpm to 800 rpm, and the air intake volume is in the range of 0.5 to 1.2 VVM.

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