Ralstonia solanacearum bacteriophage and application thereof in preparation of biological medicine for preventing and treating root-knot nematode

Abstract

The invention discloses a Ralstonia solanacearum bacteriophage and application thereof in preparation of biological medicines for preventing and treating root-knot nematodes, Ralstonia solanacearum in root-knot nematodes is taken as a target, the Ralstonia solanacearum bacteriophage is used for treating the root-knot nematodes, and the inhibition effect of the bacteriophage on the infection ability of the root-knot nematodes is determined. When the phage is used for preventing and treating the root-knot nematodes, agricultural products, people, livestock and the environment are free of pollution; particularly, the bacillus subtilis has a good application and development prospect in prevention and control of root-knot nematode of a worldwide soil-borne disease.

Description

Technical Field

[0001] The present invention belongs to the field of biological pesticides, and specifically relates to a Ralstonia solanacearum bacteriophage and its application in the preparation of biological medicines for preventing and controlling root-knot nematodes. Background Art

[0002] As a soil-borne plant disease that is prevalent and difficult to control worldwide, plant-parasitic nematodes (PPN) cause extremely serious direct damage and have become the second largest plant disease (Bird and Koltai 2000). Plant-parasitic nematodes are widely distributed throughout the world, with more than 4,100 species reported so far (Jones, Haegeman et al. 2013), causing direct economic losses of up to $157 billion each year (Abad, Gouzy et al. 2008). Among them, the most widely distributed and most harmful plant endoparasitic nematode is the root-knot nematode (Meloidogyne spp.), which has become the first of the 10 common plant pathogenic nematodes (Jones, Haegeman et al. 2013). At present, more than 100 species of root-knot nematodes have been identified worldwide (Bernard, Egnin et al. 2017), and among them, the southern root-knot nematode has the widest distribution and causes the greatest economic losses (Huang, Allen et al. 2006). The southern root-knot nematode can parasitize more than 3,000 crops (Perry and Moens 2013). Plants with more serious nematode diseases show short growth and curled leaves. Many researchers attribute this to insufficient root nutrition and water supply (Perry, Moens et al. 2009). Root-knot nematodes grow and reproduce rapidly with the planting of crops, and the density of nematodes in the soil increases sharply, eventually resulting in lower crop yields from one generation to the next. This phenomenon is more common in modern greenhouse agricultural production, and root-knot nematode disease is often accompanied by fungal diseases, which has seriously restricted the rapid modernization of agriculture (Su, Ruan et al. 2015).

[0003] At present, the methods for controlling root-knot nematodes include plant quarantine, screening of resistant varieties, agricultural control, chemical control and biological control. Plant quarantine is effective for a large number of invasive nematodes, but root-knot nematodes have a wide range of host adaptability, and the control effect is often poor and lacks substantive significance; screening of insect-resistant plants mainly uses resistance genes to resist nematode diseases. Although it can achieve disease prevention and increase production, it also has some problems, including incompatibility of distant hybridization, long breeding cycle and inability to stably express resistance genes, making it difficult to implement (Peng Deliang and Tang Wenhua 2001); agricultural control is mainly some agricultural technical measures, including crop rotation and sun-drying soil, etc. There are fewer crops that can be planted in crop rotation, especially for southern root-knot nematodes with a wide range of host adaptability; chemical control can have a significant control effect, but it causes serious pollution to food safety, the environment and the human body, and is easy to cause drug resistance (Chen Pinsan 2001), and has gradually been banned in most areas; biological control microorganisms are the most studied in biological control, mainly including bacteria, fungi and actinomycetes. Pasteuria penetrans is a biocontrol bacterium, but because it is a parasite, it is difficult to achieve large-scale cultivation, so the cost of the preparation is relatively high (Schmidt 2003). Another type of actinomycetes that are effective in controlling root-knot nematodes is actinomycetes. Their secondary metabolites can kill second-instar larvae and inhibit the hatching of eggs, and can inhibit the synthesis of RNA (Takatsu, Horiuchi et al. 2003). Avermectin is one of them. In addition, there are nematicidal fungi, which are widely distributed. When predatory fungi encounter nematodes, they change from saprophytic to parasitic. The hyphae form a contractile ring or a three-dimensional bacterial net to tightly grasp the nematodes. Then the hyphae pass through the surface of the nematode and enter the body to feed (Liu Xingzhong). Endoparasitic fungi invade the nematode body, grow in the body with the help of the host nematode's nutrition supply, and finally kill the nematode ( Urek et al. 2013). However, due to the antibacterial effect of soil, predatory and parasitic fungi cannot achieve effective control effects (Singh 2019). Since the biological control strategy of root-knot nematodes was proposed, many biological control agents have been on the market, but unstable control effects are still a common problem of biological control agents for root-knot nematodes ( Urek et al. 2013). Since all current control methods have limitations, exploring effective targets is a practical problem that needs to be solved urgently for biological control of root-knot nematodes.

[0004] Endophytic bacteria of insect pathogenic nematodes affect the pathogenicity of pathogenic nematodes (Machado, et al. 2020), endosymbiotic bacteria of pine wood nematodes are also closely related to the pathogenicity of pine wood nematodes (He, Wu et al. 2016), and endosymbiotic bacteria Wolbachia spp. have been found in plant pathogenic nematodes such as yam root rot nematodes and banana boring nematodes. They can control the characteristics of the host through cytoplasmic incompatibility. For example, some Wolbachia strains can control the population through female sterilization (Brown 2018). Therefore, endosymbiotic bacteria may be a potential target for biological control of root-knot nematodes.

[0005] Bacteriophages are viruses of bacteria, whose host range is generally narrower than the antibacterial spectrum of antibiotics. They can invade bacteria in a host-specific manner (Barrangou, Yoon et al. 2002, McLaughlin, Balaa et al. 2006, Holmfeldt, Middelboe et al. 2007), causing lysis of the host bacteria and an increase in the phage itself.

[0006] Based on the reports that plant parasitic nematodes contain endosymbiotic bacteria, this study isolated and purified Ralstonia insidiosa from southern root-knot nematodes. This is the first time that the present invention uses the endosymbiotic bacteria of root-knot nematodes, Ralstonia insidiosa, as the target bacteria to explore the effect of Ralstonia insidiosa phage on the pathogenicity of root-knot nematodes. Summary of the invention

[0007] The purpose of the present invention is to develop a green biological pesticide which is efficient, safe and highly specific, and to provide a Ralstonia solanacearum phage and its application.

[0008] The present invention isolates and purifies a bacterium from a root-knot nematode that has been disinfected on the body surface and treated with nuclease, clones a 1465 bp fragment using a 16S rDNA full-length primer, and identifies the bacterium as Ralstonia insidiosa. The strain has been deposited in the China Center for Type Culture Collection on August 24, 2022; the depository address is Wuhan University, Wuhan, Hubei, China; the deposit number is CCTCC NO: M 20221290, and its DNA sequence is SEQ ID NO:1; after confirming the presence of Ralstonia solanacearum in the root-knot nematode, a method for treating the root-knot nematode with a Ralstonia solanacearum phage using Ralstonia solanacearum as a target is provided, i.e., the use of the Ralstonia solanacearum phage in the preparation of a biological drug for the prevention and treatment of the root-knot nematode is provided, and the phage has been deposited in the General Microbiology Center of the China National Microbiological Culture Collection Administration on May 26, 2022; the depository address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; the deposit number is CGMCC No.45177, and its DNA sequence is shown in SEQ ID NO:2;

[0009] The specific technical solutions are as follows:

[0010] A Ralstonia solanacearum phage, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection on May 26, 2022, with the deposit number CGMCC No.45177.

[0011] The use of a Ralstonia solanacearum phage in the preparation of a biological drug for preventing and controlling root-knot nematodes, wherein the Ralstonia solanacearum phage is a Ralstonia solanacearum phage as claimed in claim 1.

[0012] Furthermore, the root-knot nematodes include southern root-knot nematodes, Java root-knot nematodes or peanut root-knot nematodes.

[0013] The invention discloses an application of a Ralstonia solanacearum bacteriophage in the preparation of a root-knot nematode insecticide or a root-knot nematode infection inhibitor, wherein the active ingredient of the root-knot nematode insecticide or the root-knot nematode infection inhibitor is the Ralstonia solanacearum bacteriophage.

[0014] Furthermore, the use of the Ralstonia solanacearum phage in the preparation of a root-knot nematode insecticide or a root-knot nematode infection inhibitor, wherein the minimum effective concentration of the Ralstonia solanacearum phage in the root-knot nematode insecticide or the root-knot nematode infection inhibitor is 10 8 pfu / ml.

[0015] The invention discloses an application of a Ralstonia solanacearum bacteriophage in the preparation of a biological medicine for inhibiting the infection ability of second-instar larvae of a root-knot nematode. The active ingredient of the biological medicine for inhibiting the infection ability of second-instar larvae of a root-knot nematode is the Ralstonia solanacearum bacteriophage.

[0016] The use of Ralstonia solanacearum bacteriophage in the preparation of biological medicines for inhibiting the infection ability of second-instar larvae of root-knot nematodes, wherein the minimum effective concentration of Ralstonia solanacearum bacteriophage in the biological medicines for inhibiting the infection ability of second-instar larvae of root-knot nematodes is 10 8 pfu / ml.

[0017] The working principle of the present invention is introduced as follows: after confirming the presence of Ralstonia solanacearum in the body of the root-knot nematode, the root-knot nematode is treated with Ralstonia solanacearum bacteriophage using Ralstonia solanacearum as the target. Since the second-instar larvae of the root-knot nematode are the only stage with the ability to infect, but are in a non-feeding state; although the second-instar larvae have a stylet, the inner diameter of the stylet is larger than the particle size of the bacteriophage. Therefore, this study uses passive feeding and active feeding to allow the Ralstonia solanacearum bacteriophage to enter the second-instar larvae to inhibit the infection ability of the root-knot nematode. The minimum effective concentration of the Ralstonia solanacearum bacteriophage is 10 8 pfu / ml. As the phage concentration increases, the biopharmaceutical prepared by the phage has a stronger inhibitory ability against root-knot nematodes.

[0018] Compared with the prior art, the present invention has the following beneficial effects: root-knot nematodes are treated with Ralstonia solanacearum phages, the phages have a significant inhibitory effect on the infection ability of root-knot nematodes, and the effect is fast and high in prevention efficiency; there is no pollution to agricultural products, humans, animals and the environment; in particular, the present invention has good application and development prospects for the prevention and control of root-knot nematodes, a global soil-borne disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the effect of actively feeding on Ralstonia solanacearum phage on the infection ability of the second-instar larvae of southern root-knot nematode in Experiment 1;

[0020] Figure 2 This is the effect of actively feeding on Ralstonia solanacearum phage on the infection ability of the second-instar larvae of the Javan root-knot nematode in Experiment 2;

[0021] Figure 3 This is the effect of actively feeding on Ralstonia solanacearum phage on the infection ability of the second-instar larvae of peanut root-knot nematode in Experiment 3;

[0022] Figure 4 This is the effect of passive feeding of Ralstonia solanacearum phage on the infection ability of the second-instar larvae of southern root-knot nematode in Experiment 4;

[0023] Figure 5 This is the effect of passive feeding of Ralstonia solanacearum phage on the infection ability of second-instar larvae of Javan root-knot nematode in Experiment 5;

[0024] Figure 6 This is a diagram showing the effect of passive feeding of Ralstonia solanacearum phage on the infection ability of second-instar larvae of peanut root-knot nematode in Experiment 6. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings. It should be understood that these descriptions are only exemplary and are not intended to limit the scope of the present invention. In addition, in the following invention, the description of well-known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention.

[0026] Example 1: Isolation and purification of bacteriophage

[0027] (1) Preparation of host bacteria: After repeated streaking and purification of Ralstonia solanacearum, a single colony was picked and inoculated into 50 mL of NA liquid culture medium, and cultured at 28°C and 120 rpm for 12 h for later use.

[0028] (2) Preparation of soil suspension: Add the collected soil sample into a conical flask at a ratio of 20 g soil sample per 100 mL SM suspension and shake overnight. Take it out and let it stand for more than 24 h.

[0029] (3) Enrichment and separation of bacteriophages: Filter the soil suspension through a 0.22 μm filter membrane, take 5 mL and add it to the logarithmic growth phase Ralstonia solanacearum solution, shake and culture for 12 hours, filter the co-culture solution through a 0.22 μm filter membrane to obtain the phage stock solution. Mix 0.5 mL of the phage stock solution with 0.5 mL of the logarithmic growth phase Ralstonia solanacearum solution, place it at room temperature for 15 minutes to allow the phage to adsorb to the Ralstonia solanacearum, then mix it with 6 mL of NA semi-solid culture medium cooled to about 50°C, and immediately pour it onto the solidified NA solid culture medium plate to make a double-layer plate. After the upper layer of culture medium solidifies, invert and culture it at 28°C for 24-48 hours to observe whether there are plaques.

[0030] (4) Purification of phage: When plaques appear, pick the largest single plaque and inoculate it into the bacterial solution of Ralstonia solanacearum in the logarithmic growth phase, shake it well and let it stand for 15 minutes, then put it into a shaker, and culture it at 28°C, 120r / min for 12 hours to proliferate the phage. After the proliferation culture, filter the co-culture solution with a 0.22μm filter membrane, take 0.5mL of the filtrate and mix it with 0.5mL of the bacterial solution of Ralstonia solanacearum in the logarithmic growth phase, and make a double-layer plate according to step (3), and culture it upside down at 28°C for 24-48 hours to observe whether there are plaques. Repeat the above steps 3-5 times to obtain a relatively pure phage.

[0031] (5) Prepare a Ralstonia solanacearum phage solution by diluting the phage obtained and purified in step (4) to 10% with sterile NA culture medium. 10 pfu, and keep at 4℃ for future use.

[0032] Example 2: Specific steps for determining the inhibitory effect of the above-mentioned bacteriophage on the infectivity of root-knot nematodes:

[0033] 1. Obtaining tomato seedlings planted in Pluronic colloids

[0034] Soak the tomato seeds in a 9 cm culture plate filled with warm water (boiling water: cold water = 3:2), so that the warm water submerges the tomato seeds and prevents the tomato seeds from clumping, and place the plate in a constant temperature box at 28°C for 24 hours. After 24 hours, take out the plate and wash the tomato seeds 10 times with sterile water. Use sterile tweezers to spread the filter paper on the 9 cm culture plate and add 3 ml of sterile water to it. After being fully washed with sterile water, the tomato seeds are dotted one by one on the moistened filter paper and placed in a constant temperature box at 28°C for 72 hours. After 72 hours, the tomato seedlings with a root length of 0.5 cm are inoculated into a cell culture plate (6 wells) containing 2 ml of 23% Pluronic solution. Each well is inoculated with 6 tomato seedlings, and 6 replicates are set.

[0035] 2. Root-knot nematodes feed on bacteriophages

[0036] The second-instar larvae of the root-knot nematode are the only stage that has the ability to infect. Although the second-instar larvae have a stylet, the inner diameter of the stylet is larger than the particle size of the phage, so the phage can enter the root-knot nematode. Therefore, this study used two methods to allow the phage to enter the second-instar larvae, namely, the immersion method to make the nematodes actively feed on the phage and the carbachol-stimulated second-instar larvae to passively feed on the phage.

[0037] 2.1 Root-knot nematodes actively feed on bacteriophages

[0038] Collect freshly hatched second-instar larvae, prepare a nematode suspension with 1 / 4 M9 buffer, and divide the nematode suspension into two equal parts. Add an appropriate amount of NA culture solution (g / L: 10 g peptone, 3 g beef extract, 5 g sodium chloride, natural pH) to the first part to make its concentration 1 / 10 times that of the original solution, and set it as a blank control. Add an equal volume of Ralstonia solanacearum phage solution (Ralstonia solanacearum phage suspended in NA culture solution) to the second nematode suspension to make the final concentration of phage reach 10 8 The nematode suspension was placed on a rotary shaker at 28°C for 24 hours. After 24 hours, the nematodes were washed five times with 1 / 4 M9 buffer and prepared into a nematode suspension with a concentration of 50 nematodes / μl.

[0039] 2.2 Passive feeding of phages by root-knot nematodes

[0040] Freshly hatched second-instar larvae were collected and placed in stimulation buffer (g / L: 5.8 g Na 2 HPO 4 .7H 2 O, 3 g KH 2 PO 4 , 5 g NaCl, 0.44 g spermidine, 0.5 g gelatin, pH natural), placed on a rotary shaker at room temperature (speed of 10 rpm) for 12 hours. After 12 hours, the nematodes were thoroughly washed 10 times with 1 / 4 M9 buffer, and a nematode suspension was prepared, which was then divided into two equal parts. An appropriate amount of NA culture solution was added to the first portion to make its concentration 1 / 10 times that of the original solution, which was set as a blank control. An equal volume of Ralstonia solanacearum phage solution (Ralstonia solanacearum phage was suspended in NA culture solution) was added to the second nematode suspension to make the final concentration of the phage reach 10 8 The nematode suspension was placed on a rotary shaker and rotated at room temperature (10 rpm) for 24 hours. After 24 hours, the nematodes were washed 5 times with 1 / 4 M9 buffer and prepared into a nematode suspension with a concentration of 50 nematodes / μl.

[0041] 3. Detection of infection ability of second-instar larvae

[0042] To clarify the effect of Ralstonia solanacearum phage treatment on inhibiting the infectivity of root-knot nematodes, this study specifically quantified the changes in the infectivity of root-knot nematodes caused by phage treatment, namely, the infectivity of tomato seedlings colonized in Pluronic colloids (23%).

[0043] The second-instar larvae of root-knot nematodes treated with NA culture medium and Ralstonia solanacearum phage were inoculated into the root tips of seedlings colonized in Pluronic colloids, with 100 nematodes inoculated within 0.5 cm of the periphery of each root tip. Incubate at 28°C in the dark for 48 hours. After 48 hours, place the cell culture plate on ice for 10 minutes. After 10 minutes, move the tomato seedlings to a 5% sodium hypochlorite solution, let stand for 30 minutes, and rinse with sterile water until odorless. Place the tomato seedlings in a beaker containing 5 ml of acid fuchsin solution and heat in a boiling water bath for 25 minutes until the tomato seedlings are completely colored. Prepare slides and observe under a microscope. Count the number of nematodes in the roots of each treated tomato, and use the following formula to calculate the nematode infectivity:

[0044] Relative infection rate (%) = A / B*100

[0045] Infection inhibition rate (%) = (BA) / B*100

[0046] Among them: A is the number of root-knot nematodes that infect and enter the roots of the treatment group, and B is the number of root-knot nematodes that infect and enter the roots of the control group.

[0047] Embodiment 3:

[0048] Experiment 1: The operation of the second-instar larvae of southern root-knot nematode actively feeding on bacteriophages and the statistics of infection results

[0049] Freshly hatched second-instar larvae of southern root-knot nematodes were collected and a nematode suspension was prepared with 1 / 4 M9 buffer, and the nematode suspension was divided into two portions. An appropriate amount of NA culture solution was added to the first portion to make its concentration 1 / 10 of the original solution, which was set as a blank control. An equal volume of Ralstonia solanacearum phage solution (Ralstonia solanacearum phage suspended in NA culture solution) was added to the second nematode suspension to make the final concentration of phage reach 10 8 pfu / ml. The above nematode suspension was placed on a rotary shaker and placed at 28°C for 24 hours. After 24 hours, the nematodes were fully washed 5 times with 1 / 4 times M9 buffer and prepared into a nematode suspension with a concentration of 150 / μl. The nematode suspensions treated with NA culture solution and Ralstonia solanacearum phage were inoculated into the root tips of seedlings colonized in Pluronic colloids, and the nematode inoculation amount within 0.5 cm of the periphery of each root tip was 100. Stand for 48 hours under dark conditions at 28°C. After 48 hours, the cell culture plate was placed on ice for 10 minutes. After 10 minutes, the tomato seedlings were moved to 5% sodium hypochlorite solution, stood for 30 minutes, and rinsed with sterile water until odorless. Stain with acid fuchsin solution, prepare slides and observe under a microscope, count the number of nematodes in the roots of each treated tomato, and determine the ability of Ralstonia solanacearum phage to inhibit the infection of southern root-knot nematodes.

[0050] The results are as follows Figure 1 As shown, in the absence of nerve stimulants, after the second-instar larvae of the southern root-knot nematode ingested the Ralstonia solanacearum phage, their ability to infect the host tomato plant was significantly reduced (A), and the infection inhibition rate reached 47.05% (B).

[0051] Experiment 2: The operation of the second-instar larvae of Javan root-knot nematode actively feeding on bacteriophages and the statistics of infection results

[0052] Freshly hatched second-instar larvae of Javan root-knot nematodes were collected and a nematode suspension was prepared with 1 / 4 M9 buffer, and the nematode suspension was divided into two portions. An appropriate amount of NA culture solution was added to the first portion to make its concentration 1 / 10 of the original solution, which was set as a blank control. An equal volume of Ralstonia solanacearum phage solution (Ralstonia solanacearum phage suspended in NA culture solution) was added to the second portion of the nematode suspension to make the final concentration of the phage reach 10 8pfu / ml. The above nematode suspension was placed on a rotary shaker and placed at 28°C for 24 hours. After 24 hours, the nematodes were fully washed 5 times with 1 / 4 M9 buffer and prepared into a nematode suspension with a concentration of 150 / μl. The nematode suspensions treated with NA culture solution and Ralstonia solanacearum phage were inoculated into the root tips of seedlings colonized in Pluronic colloids, and the nematode inoculation amount within 0.5 cm of the periphery of each root tip was 100. Stand for 48 hours under dark conditions at 28°C. After 48 hours, the cell culture plate was placed on ice for 10 minutes. After 10 minutes, the tomato seedlings were moved to 5% sodium hypochlorite solution, stood for 30 minutes, and rinsed with sterile water until odorless. Stain with acid fuchsin solution, prepare slides and observe under a microscope, count the number of nematodes in the roots of each treated tomato, and determine the situation of Ralstonia solanacearum phage reducing the infection ability of Java root-knot nematodes.

[0053] The results are as follows Figure 2 As shown, in the absence of nerve stimulants, after the second-instar larvae of the Javan root-knot nematode ingested the Ralstonia solanacearum phage, their ability to infect the host tomato plant was significantly reduced (A), and the infection inhibition rate reached 52.94% (B).

[0054] Experiment 3: Operation of second-instar larvae of peanut root-knot nematode actively feeding on bacteriophages and statistics of infection results

[0055] Freshly hatched second-instar larvae of peanut root-knot nematodes were collected and a nematode suspension was prepared with 1 / 4 M9 buffer, and the nematode suspension was divided into two portions. An appropriate amount of NA culture solution was added to the first portion to make its concentration 1 / 10 of the original solution, which was set as a blank control. An equal volume of Ralstonia solanacearum phage solution (Ralstonia solanacearum phage suspended in NA culture solution) was added to the second portion of the nematode suspension to make the final concentration of the phage reach 10 8 pfu / ml. The above nematode suspension was placed on a rotary shaker and placed at 28°C for 24 hours. After 24 hours, the nematodes were fully washed 5 times with 1 / 4 times M9 buffer and prepared into a nematode suspension with a concentration of 150 / μl. The nematode suspensions treated with NA culture solution and Ralstonia solanacearum phage were inoculated into the root tips of seedlings colonized in Pluronic colloids, and the nematode inoculation amount within 0.5 cm of the periphery of each root tip was 100. Stand for 48 hours under dark conditions at 28°C. After 48 hours, the cell culture plate was placed on ice for 10 minutes. After 10 minutes, the tomato seedlings were moved to 5% sodium hypochlorite solution, stood for 30 minutes, and rinsed with sterile water until odorless. Stain with acid fuchsin solution, prepare slides and observe under a microscope, count the number of nematodes in the roots of each treated tomato, and determine the situation of Ralstonia solanacearum phage reducing the infection ability of peanut root-knot nematodes.

[0056] The results are as follows Figure 3As shown, in the absence of nerve stimulants, after the second-instar larvae of peanut root-knot nematodes ingested the Ralstonia solanacearum phage, their ability to infect the host tomato plants was significantly reduced (A), and the infection inhibition rate reached 54.55% (B).

[0057] Experiment 4: The passive feeding of second-instar larvae of southern root-knot nematode on bacteriophages and the statistics of infection results

[0058] Freshly hatched second-instar larvae of southern root-knot nematodes were collected and added to a buffer solution containing 0.5 mg / ml carbachol, and placed on a rotary shaker at room temperature (speed of 10 rpm) for 12 hours. After 12 hours, the nematodes were thoroughly washed 10 times with 1 / 4 M9 buffer, and a nematode suspension was prepared, which was then divided into two equal parts. An appropriate amount of NA culture solution was added to the first portion to make its concentration 1 / 10 times that of the original solution, which was set as a blank control. An equal volume of Ralstonia solanacearum phage solution (Ralstonia solanacearum phage was suspended in NA culture solution) was added to the second nematode suspension to make the final concentration of the phage reach 10 8 pfu / ml. The above nematode suspension was placed on a rotary shaker and rotated at room temperature (10rpm) for 24 hours. After 24 hours, the nematodes were fully washed 5 times with 1 / 4 times M9 buffer and prepared into a nematode suspension with a concentration of 150 / μl. The nematodes treated with NA culture solution and Ralstonia solanacearum phage were inoculated into the root tips of seedlings colonized in Pluronic colloids, and the nematode inoculation amount within 0.5 cm of the periphery of each root tip was 100. Stand for 48 hours under 28°C dark conditions. After 48 hours, the cell culture plate was placed on ice for 10 minutes. After 10 minutes, the tomato seedlings were moved to 5% sodium hypochlorite solution, stood for 30 minutes, and rinsed with sterile water until tasteless. Stain with acid fuchsin solution, prepare slides and observe under a microscope, count the number of southern root-knot nematodes in the roots of each treated tomato, and determine the ability of Ralstonia solanacearum phage to reduce the infection ability of southern root-knot nematodes.

[0059] The results are as follows Figure 4 As shown in the figure, after stimulation by nerve stimulants, the second-instar larvae of southern root-knot nematodes ingested the Ralstonia solanacearum phage, and their ability to infect host tomato plants was significantly reduced (A), and the infection inhibition rate reached 39.45 (B). The above experimental results show that the infectivity of the second-instar larvae is significantly reduced after feeding on the Ralstonia solanacearum phage, indicating that the dangerous Ralstonia promotes the infection of the second-instar larvae on the host plant.

[0060] Experiment 5: The passive feeding of phages by the second-instar larvae of the Javan root-knot nematode and the statistics of infection results

[0061] Freshly hatched second-instar larvae of the Javan root-knot nematode were collected and added to a buffer solution containing 0.5 mg / ml carbachol, and placed on a rotary shaker at room temperature (speed of 10 rpm) for 12 hours. After 12 hours, the nematodes were thoroughly washed 10 times with 1 / 4 M9 buffer, and a nematode suspension was prepared, which was then divided into two equal parts. An appropriate amount of NA culture solution was added to the first portion to make its concentration 1 / 10 times that of the original solution, which was set as a blank control. An equal volume of Ralstonia solanacearum phage solution (Ralstonia solanacearum phage was suspended in NA culture solution) was added to the second nematode suspension to make the final concentration of the phage reach 10 8 pfu / ml. The above nematode suspension was placed on a rotary shaker and rotated at room temperature (10rpm) for 24 hours. After 24 hours, the nematodes were fully washed 5 times with 1 / 4 times M9 buffer and prepared into a nematode suspension with a concentration of 100 / μl. The nematodes treated with NA culture solution and Ralstonia solanacearum phage were inoculated into the root tips of seedlings colonized in Pluronic colloids, and the nematode inoculation amount within 0.5 cm of the periphery of each root tip was 200. Stand for 48 hours under 28°C dark conditions. After 48 hours, the cell culture plate was placed on ice for 10 minutes. After 10 minutes, the tomato seedlings were moved to 5% sodium hypochlorite solution, stood for 30 minutes, and rinsed with sterile water until tasteless. Stain with acid fuchsin solution, prepare slides and observe under a microscope, count the number of southern root-knot nematodes in the roots of each treated tomato, and determine the ability of Ralstonia solanacearum phage to reduce the infection ability of Java root-knot nematodes.

[0062] The results are as follows Figure 5 As shown, after being stimulated by nerve irritants, the second-instar larvae of the Javan root-knot nematode ingested the Ralstonia solanacearum phage, and their ability to infect the host tomato plant was significantly reduced (A), and the infection inhibition rate reached 54.82% (B).

[0063] Experiment 6: Operation of passive feeding of phage by second-instar larvae of peanut root-knot nematode and statistics of infection results

[0064] Collect freshly hatched second-instar larvae of peanut root-knot nematodes, add them to a buffer solution containing 0.5 mg / ml carbachol, and place them on a rotary shaker at room temperature (speed of 10 rpm) for 12 hours. After 12 hours, wash the nematodes 10 times with 1 / 4 M9 buffer, and make a nematode suspension, which is then divided into two equal parts. Add an appropriate amount of NA culture solution to the first part so that its concentration is 1 / 10 times that of the original solution, and set it as a blank control. Add an equal volume of Ralstonia solanacearum phage solution to the second nematode suspension (Ralstonia solanacearum phage is suspended in NA culture solution) to make the final concentration of the phage reach 10 8pfu / ml. The above nematode suspension was placed on a rotary shaker and rotated at room temperature (10rpm) for 24 hours. After 24 hours, the nematodes were fully washed 5 times with 1 / 4 times M9 buffer and prepared into a nematode suspension with a concentration of 100 / μl. The nematodes treated with NA culture solution and Ralstonia solanacearum phage were inoculated into the root tips of seedlings colonized in Pluronic colloids, and the nematode inoculation amount within 0.5 cm of the periphery of each root tip was 200. Stand for 48 hours under 28°C dark conditions. After 48 hours, the cell culture plate was placed on ice for 10 minutes. After 10 minutes, the tomato seedlings were moved to 5% sodium hypochlorite solution, stood for 30 minutes, and rinsed with sterile water until tasteless. Stain with acid fuchsin solution, prepare slides and observe under a microscope, count the number of southern root-knot nematodes in the roots of each treated tomato, and determine the ability of Ralstonia solanacearum phage to reduce the infection ability of Java root-knot nematodes.

[0065] The results are as follows Figure 6 As shown, after being stimulated by nerve irritants, the second-instar larvae of peanut root-knot nematodes ingested the Ralstonia solanacearum phage, and their ability to infect the host tomato plants was significantly reduced (A), and the infection inhibition rate reached 57.26% (B).

[0066] It should be understood that the above specific embodiments of the present invention are only used for illustrative inventions or to explain the principles of the present invention, and do not constitute limitations on the present invention. Therefore, any modifications, equivalent substitutions, improvements, etc. made without departing from the spirit and scope of the present invention should be included in the protection scope of the present invention. In addition, the appended claims of the present invention are intended to cover all changes and modifications that fall within the scope and boundaries of the appended claims, or the equivalent forms of such scope and boundaries.

[0067] SEQ ID NO:1

[0068]

[0069]

[0070] SEQ ID NO:2

[0071]

[0072]

Claims

1. A Ralstonia solanacearum phage, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on May 26, 2022, with the deposit number CGMCC No.45177.

2. Use of a Ralstonia solanacearum phage in the preparation of a biological drug for controlling root-knot nematodes, wherein the Ralstonia solanacearum phage is a Ralstonia solanacearum phage as claimed in claim 1.

3. The use according to claim 2, It is characterized in that The root-knot nematodes include southern root-knot nematodes, Java root-knot nematodes or peanut root-knot nematodes.

4. Use of Ralstonia solanacearum phage in the preparation of root-knot nematode insecticides or root-knot nematode infection inhibitors, Features: The active ingredient of the root-knot nematode insecticide or root-knot nematode infection inhibitor is the Ralstonia solanacearum phage as claimed in claim 1.

5. Use of the Ralstonia solanacearum phage according to claim 4 in the preparation of a root-knot nematode insecticide or a root-knot nematode infection inhibitor, It is characterized in that The minimum effective concentration of Ralstonia solanacearum phage in root-knot nematode insecticides or root-knot nematode infestation inhibitors is 10 8 pfu / ml.

6. Application of Ralstonia solanacearum phage in the preparation of biological drugs for inhibiting the infection ability of second-instar larvae of root-knot nematodes, Features: The active ingredient of the biological drug for inhibiting the infection ability of the second-instar larvae of the root-knot nematode is the Ralstonia solanacearum phage as described in claim 1.

7. Use of the Ralstonia solanacearum phage according to claim 6 in the preparation of a biological drug for inhibiting the infection ability of second-instar larvae of root-knot nematodes, It is characterized in that The minimum effective concentration of Ralstonia solanacearum phage in the biological drug that inhibits the infectivity of the second-instar larvae of the root-knot nematode is 10 8 pfu / ml.

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