A method for batch inoculation of phages to control tobacco bacterial wilt

Abstract

The present invention discloses a method for batch inoculation of phages to control tobacco bacterial wilt, including: Step S1, culturing phages and Ralstonia solanacearum together to obtain phages with the ability to infect and lyse tobacco Ralstonia solanacearum; Step S2, encapsulating the phages into a nano-carrier to prepare nanoparticles, where the nano-carrier is a biodegradable nano-carrier; Step S3, cutting off water for hardening seedlings 5 - 7 days before transplanting flue-cured tobacco floating seedlings. After the hardening of seedlings is completed, put 2 - 3 cm of shallow water in the seedling-raising pool, add the phage-containing nanoparticles and an adhesive to the nutrient pool, and put the floating seedling trays back into the seedling-raising pool for inoculation. By combining phage therapy and nanotechnology, the present invention adds nano-encapsulated phages to tobacco floating seedling raising, which can not only inoculate a large number of tobacco seedlings more conveniently and quickly, but also extend the persistence of phages in the soil and improve their control efficacy.

Description

Technical Field

[0001] The present invention relates to a method for batch inoculating phages to control tobacco bacterial wilt, belonging to the technical field of inoculating phages to control tobacco bacterial wilt. Background Art

[0002] Plant bacterial wilt is a systemic disease caused by the infection of Ralstonia solanacearum. It is a typical vascular bundle disease, and the most significant symptom is wilting. When the disease occurs severely, it will cause the death of the whole plant. The occurrence of tobacco bacterial wilt is the result of the combined action of tobacco, pathogenic bacteria, and the environment. Environmental temperature, humidity, soil pH, and the population of Ralstonia solanacearum in the soil are all closely related to the occurrence of tobacco bacterial wilt. At present, although comprehensive measures such as chemical control, rational crop rotation, planting disease-resistant varieties, adjusting soil microorganisms, and adjusting the tobacco seedling transplanting period are adopted in production, the occurrence of this disease still cannot be effectively prevented. Therefore, people expect to develop safer and more effective methods and strategies for controlling bacterial wilt.

[0003] With the improvement of people's requirements for environmental safety and food safety, with the widespread use of antibiotics, and the emergence of drug resistance or antibiotic resistance, phage therapy is highly expected as an alternative to traditional drug therapy. Phages can rapidly proliferate in sensitive host bacteria and cause their lysis. This characteristic endows phages with the ability to be natural "killers" of pathogenic bacteria and also has application potential in biological control. However, in the control of crop bacterial wilt, the use of phages infecting Ralstonia solanacearum to control bacterial wilt is restricted to a certain extent by the inoculation method of phages infecting Ralstonia solanacearum. Currently, the main methods for inoculating phages of Ralstonia solanacearum in tobacco plants are the root irrigation method and the root dipping method. Among them, root irrigation is an operation of inoculating one by one, and root dipping is to put plant seedlings into the inoculation liquid before planting. This method can achieve batch operation, but the inoculated cell concentration is relatively low, and the effect is inferior to root irrigation. Summary of the Invention

[0004] Based on the above, the present invention provides a method for batch inoculating phages with a high inoculated cell concentration to control tobacco bacterial wilt to overcome the deficiencies of the prior art.

[0005] The technical solution of the present invention is: A method for batch inoculating phages to control tobacco bacterial wilt, comprising:

[0006] Step S1, phage acquisition: Culturing phages and Ralstonia solanacearum together to obtain phages with the ability to infect and lyse tobacco Ralstonia solanacearum;

[0007] Step S2, phage encapsulation: Encapsulating phages into a nano-carrier to prepare nanoparticles, and the nano-carrier is a biodegradable nano-carrier;

[0008] Step S3, Phage Inoculation: Cut off the water supply for 5 - 7 days before transplanting the tobacco seedlings grown by floating system for hardening. After the hardening is completed, add 2 - 3 cm of shallow water to the seedling nursery pond. Add the phage-containing nanoparticles and the adhesive to the nutrient pond, and then place the floating seedling trays back into the seedling nursery pond for inoculation.

[0009] As a further improvement of the present invention, in step S1, the method for obtaining phages is as follows: In a sterilized Erlenmeyer flask, add CPG liquid medium and sterile water containing Ralstonia solanacearum, shake and culture at 28°C for 2 - 4 h until the absorbance OD600 is between about 0.2 - 0.3, then add 100 - 300 μl of sterile water containing phages, and continue to shake and culture at 28°C for 2 - 3 h; then centrifuge the culture solution at a speed of 8000 r / min for 10 min, take the supernatant, and filter it through a 0.22 μm filter membrane to obtain a phage solution.

[0010] As a further improvement of the present invention, in step S2, the method for encapsulating phages is as follows:

[0011] S21, Slowly add the phage solution to the nano-carrier solution, and at the same time use a high-speed homogenizer or an ultrasonic emulsifier for emulsification to form an oil-in-water emulsion;

[0012] S22, Add the oil-in-water emulsion to water and perform emulsification again to form an oil-in-water-in-water emulsion;

[0013] S23, By means of stirring and reducing the temperature, slowly volatilize dichloromethane to form solidified nanoparticles;

[0014] S24, Use a centrifuge to collect the nanoparticles, and then wash them with sterile water or physiological saline to remove the residual organic solvents on the surface;

[0015] S25, Freeze-dry the washed nanoparticles.

[0016] As a further improvement of the present invention, the nano-carrier is a poly(lactic-co-glycolic acid) copolymer, and the poly(lactic-co-glycolic acid) copolymer powder is dissolved in dichloromethane to form a solution.

[0017] As a further improvement of the present invention, the adhesive is sodium alginate or xanthan gum.

[0018] Advantages of the present invention: By combining phage therapy and nanotechnology, the present invention provides a safer, more effective and environmentally friendly method for preventing and controlling tobacco bacterial wilt. Encapsulating phages into nanocarriers can significantly enhance the stability of phages in the external environment. This encapsulation protects phages from environmental damage, increases their persistence in the soil, and thus extends the duration of the prevention and control effect. Adding phage-containing nanoparticles to the seedling-raising pond and combining them with tobacco floating seedling raising can more conveniently and quickly process a large number of tobacco seedlings compared to the traditional root irrigation method, which is suitable for large-scale application. It not only saves time and labor, but also the encapsulated phages extend their prevention and control time and improve the prevention effect. Using an adhesive can increase the adhesion effect of encapsulated phages in the tobacco root system and the base. Phages that infect Ralstonia solanacearum are specific and only infect and lyse Ralstonia solanacearum in tobacco, and are harmless to other bacterial communities in tobacco seedlings and the soil. Detailed implementation manners

[0019] To make the above objects, features and advantages of the present invention more obvious and understandable, the following detailed description is made of the specific implementation manners of the present invention. Many specific details are set forth in the following description in order to fully understand the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific implementations disclosed below.

[0020] Example 1

[0021] A method for batch inoculating phages to prevent and control tobacco bacterial wilt, comprising:

[0022] Step S1, phage acquisition

[0023] In a sterilized 300 - 500 ml Erlenmeyer flask, add 100 - 300 ml of mCPG liquid medium and 100 - 300 μl of sterile water containing Ralstonia solanacearum, shake and culture at 28°C for 2 - 4 h, and the absorbance OD 600 is about between 0.2 - 0.3, then add 100 - 300 μl of sterile water containing phages, and continue to shake and culture at 28°C for 2 - 3 h; then centrifuge the culture solution at 8000 r / min for 10 min, take the supernatant, and filter it with a 0.22 μm filter membrane to obtain a phage solution with a concentration of 1×10 7 -1×10 9 PFU / ml. After the phage quantity reaches the standard (concentration of 1×10 7 -1×10 9 PFU / ml), it is considered that the preparation of the phage solution that infects Ralstonia solanacearum is completed.

[0024] CPG liquid medium: 1 g of hydrolyzed casein, 10 g of peptone, 5 g of glucose, 1000 mL of distilled water; pH 7.0.

[0025] Step S2, phage encapsulation

[0026] S21, Dissolve poly (lactic-co-glycolic acid) copolymer powder in dichloromethane to form a nano-carrier solution with a concentration of 10% w / v. Slowly add a phage solution with a concentration of 1×10 9 PFU / ml to the nano-carrier solution. The volume ratio of the phage solution to the nano-carrier solution is 1:1. At the same time, use a high-speed homogenizer or an ultrasonic emulsifier for emulsification to form a W / O (water-in-oil) emulsion;

[0027] S22, Add the W / O emulsion to a large amount of water. The volume ratio of the W / O emulsion to water is 1:4, and emulsify again for 10 - 15 minutes to form a W / O / W emulsion;

[0028] S23, Slowly evaporate dichloromethane by stirring and maintaining the temperature at room temperature to form solidified poly (lactic-co-glycolic acid) nanoparticles;

[0029] S24, Use a centrifuge to collect the nanoparticles at 8000 r / min for 10 minutes, and then wash twice with sterile water to remove the residual organic solvents on the surface;

[0030] S25, Freeze-dry the washed nanoparticles at -50°C to -80°C for 12 - 24 hours.

[0031] Step S3, phage inoculation

[0032] The tobacco seedlings are implemented according to the "Technical Regulations for Flue-cured Tobacco Floating Seedling Raising in Guizhou Province" (DB52 / 661 - 2010). Six days before transplanting the flue-cured tobacco floating seedlings, cut off the water for hardening the seedlings. After the hardening of the seedlings, put 3 cm of shallow water in the seedling raising pool, and put in the nanoparticles containing phages so that the concentration of phages in the seedling raising pool is 1×10 6 PFU / ml, add a certain amount of sodium alginate so that the concentration of sodium alginate in the seedling raising pool is 0.5%. Put the floating seedling tray back into the seedling raising pool for inoculation for 24 hours. When inoculating, the roots and substrates of the tobacco seedlings should be fully immersed in water.

[0033] Control 1

[0034] A method for batch inoculation of phages to control tobacco bacterial wilt, including:

[0035] Step S1, phage acquisition

[0036] In a sterilized 300 - 500 ml Erlenmeyer flask, add 100 - 300 ml of mCPG liquid medium and 100 - 300 μl of sterile water containing Ralstonia solanacearum. Incubate with shaking at 28°C for 2 - 4 h until the absorbance OD600 is between approximately 0.2 - 0.3. Then add 100 - 300 μl of sterile water containing phage, and continue to incubate with shaking at 28°C for 2 - 3 h. After that, centrifuge the culture solution at 8000 r / min for 10 min, take the supernatant, and filter it through a 0.22 μm filter membrane to obtain a phage solution with a concentration of 1×10 7 -1×10 9 PFU / ml. After the phage quantity meets the standard (concentration of 1×10 7 -1×10 9 PFU / ml), it is considered that the preparation of the phage solution for infecting Ralstonia solanacearum is completed.

[0037] CPG liquid medium: 1 g of casein hydrolysate, 10 g of peptone, 5 g of glucose, 1000 mL of distilled water; pH 7.0.

[0038] Step S2, Phage inoculation

[0039] The tobacco seedlings are cultivated according to the "Technical Regulations for Floating Seedling Raising of Flue-cured Tobacco in Guizhou Province" (DB52 / 661 - 2010). Six days before transplanting the floating seedlings of flue-cured tobacco, cut off the water for hardening the seedlings. After the hardening is completed, put 3 cm of shallow water in the seedling-raising pool, add the phage solution to make the concentration of phage in the seedling-raising pool 1×10 6 PFU / ml, and put the floating seedling tray back into the seedling-raising pool for inoculation for 24 hours. During inoculation, the roots and substrate of the tobacco seedlings should be fully immersed in water.

[0040] Comparative Example 2

[0041] Step S1, Phage acquisition

[0042] In a sterilized 300 - 500 ml Erlenmeyer flask, add 100 - 300 ml of mCPG liquid medium and 100 - 300 μl of sterile water containing Ralstonia solanacearum. Incubate with shaking at 28°C for 2 - 4 h until the absorbance OD600 is between approximately 0.2 - 0.3. Then add 100 - 300 μl of sterile water containing phage, and continue to incubate with shaking at 28°C for 2 - 3 h. After that, centrifuge the culture solution at 8000 r / min for 10 min, take the supernatant, and filter it through a 0.22 μm filter membrane to obtain a phage solution with a concentration of 1×10 7 -1×10 9 PFU / ml. After the phage quantity meets the standard (concentration of 1×10 7 -1×10 9 PFU / ml), it is considered that the preparation of the phage solution for infecting Ralstonia solanacearum is completed.

[0043] CPG liquid medium: 1 g of hydrolyzed casein, 10 g of peptone, 5 g of glucose, 1000 mL of distilled water; pH 7.0.

[0044] Step S2, phage inoculation

[0045] The tobacco seedlings are implemented according to the "Technical Regulations for Flue-cured Tobacco Float Seedling Raising in Guizhou Province" (DB52 / 661-2010). After transplanting the flue-cured tobacco, the phage concentration is diluted to 1×10 6 PFU / ml, and the roots are irrigated with 300 mL of the bacterial liquid per plant, and the phage infecting Ralstonia solanacearum is inoculated into the tobacco field soil.

[0046] The inoculation tests in Example 1 and Comparative Example 1 above were carried out in different seedling-raising ponds in the same seedling-raising greenhouse. The test tobacco seedlings were Yunyan 87. The tobacco seedlings in Example 1, Comparative Example 1, Comparative Example 2, and the non-inoculated tobacco seedlings (as the blank group) were transplanted in the same field. Soil samples around the roots of the tobacco seedlings were collected 1 day, 5 days, and 10 days after transplantation to detect the phage concentration. The phage concentration detection was divided into two types. One was the phage concentration of the substrate sample (away from the root system) after transplantation, and the other was the phage concentration of the substrate sample attached to the root system after transplantation.

[0047] The phage concentration in Comparative Example 1, Comparative Example 2, and the blank group was detected as follows: Take 0.5 g of the soil sample around the roots of the inoculated tobacco seedlings, add 10 ml of pure water, mix well, centrifuge and take the supernatant, and obtain the filtrate through a filter with a pore size of 0.22 μm. Detect the phage content in the filtrate sample. The specific method is as follows: Take 20-50 μl of the above filtrate sample and mix it evenly with 100 μl of the Ralstonia solanacearum liquid with a concentration of 1×10 7 cfu / ml, coat it on the CPG solid medium, and co-culture it at 28 °C for 16-20 h. Observe the clear phage plaques, and estimate the concentration of phages released in the soil sample according to the number of phage plaques.

[0048] The phage concentration in Example 1 was detected as follows: Take 0.5 g of the soil sample around the roots of the inoculated tobacco seedlings, add 10 ml of pure water, mix well to obtain a soil suspension, use an ultrasonic crusher to treat the soil suspension to release the nano-encapsulated phages, centrifuge and take the supernatant, and obtain the filtrate through a filter with a pore size of 0.22 μm. Detect the phage content in the filtrate sample. The specific method is as follows: Take 20-50 μl of the above filtrate sample and mix it evenly with 100 μl of the Ralstonia solanacearum liquid with a concentration of 1×10 7 cfu / ml, coat it on the CPG solid medium, and co-culture it at 28 °C for 16-20 h. Observe the clear phage plaques, and estimate the concentration of phages released in the soil sample according to the number of phage plaques.

[0049] Table 1 Phage concentration values

[0050]

[0051]

[0052] As can be seen from Table 1, by encapsulating phages into nano-carriers and adding adhesives to the inoculation solution, compared with non-encapsulated phages, it can not only greatly increase the phage concentration in the substrate samples after planting and the substrate samples attached to tobacco roots, but also maintain the persistence of phages in the soil (the decrease rate of the concentration of encapsulated phages over time is less than that of non-encapsulated phages), and the duration of prevention and control improvement is much less than that of non-encapsulated phages. Compared with the root irrigation method, although the phage concentration after inoculation is lower than that of the root irrigation method, the decrease rate of the phage concentration over time is also much less than that of the root irrigation method, and finally the phage concentration in the soil is greater than that of the root irrigation method, indicating that the inoculation method of the present invention is superior to the root irrigation method.

[0053] The above-described embodiments merely represent several implementation manners of the present invention. The description thereof is relatively specific and detailed, but it should not be construed as a limitation to the scope of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the appended claims.

Claims

1. A method for batch inoculation of phages to control tobacco bacterial wilt, characterized in that, Including: Step S1, phage acquisition: Co-culturing phages and Ralstonia solanacearum to obtain phages with the ability to infect and lyse Ralstonia solanacearum in tobacco; Step S2, phage encapsulation: Encapsulating phages into a nano-carrier to prepare nanoparticles, and the nano-carrier is a biodegradable nano-carrier; Step S3, phage inoculation: Stop watering and harden the seedlings 5 - 7 days before transplanting the floating seedlings of flue-cured tobacco. After the hardening of the seedlings, add 2 - 3 cm of shallow water to the seedling raising pool. Add the nanoparticles containing phages and an adhesive to the nutrient pool, and then put the floating seedling tray back into the seedling raising pool for inoculation. The adhesive is sodium alginate or xanthan gum; Among them, in step S2, the method of phage encapsulation is: S21, Slowly add the phage solution to the nano-carrier solution, and at the same time use a high-speed homogenizer or an ultrasonic emulsifier for emulsification to form an oil-in-water emulsion. The nano-carrier is a poly(lactic-co-glycolic acid) copolymer, and the poly(lactic-co-glycolic acid) copolymer powder is dissolved in dichloromethane to form a solution; S22, Add the oil-in-water emulsion to water and perform emulsification again to form an oil-in-water-in-water emulsion; S23, Slowly volatilize dichloromethane by stirring and reducing the temperature to form solidified nanoparticles; S24, Use a centrifuge to collect the nanoparticles, and then wash them with sterile water or physiological saline to remove the residual organic solvents on the surface; S25, Freeze-dry the washed nanoparticles.

2. The method for mass inoculation of phages to control tobacco bacterial wilt according to claim 1, characterized in that In step S1, the method of phage acquisition is: In a sterilized Erlenmeyer flask, add CPG liquid medium and sterile water containing Ralstonia solanacearum, shake and culture at 28 °C for 2 - 4 h, the absorbance OD600 is 0.2 - 0.3, then add 100 - 300 μl of sterile water containing phages, and continue to shake and culture at 28 °C for 2 - 3 h; then centrifuge the culture solution at a speed of 8000 r / min for 10 min, take the supernatant, and filter it through a 0.22 μm filter membrane to obtain a phage solution.

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