Chemical pesticide-resistant ralstonia solanacearum phage and application thereof

Abstract

The invention provides a ralstonia solanacearum bacteriophage resistant to chemical pesticides and application thereof, and relates to the technical field of biological control. The Ralstonia solanacearum phage RSP35 is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number of the Ralstonia solanacearum phage RSP35 is CCTCC M 2024498. The bacteriophage has high tolerance to chemical pesticides and strong specificity, can be used for preparing a pharmaceutical preparation for preventing and / or treating bacterial wilt and a pharmaceutical composition for preventing and / or treating bacterial wilt, and provides a feasible scheme for preventing and treating bacterial wilt.

Description

Technical Field

[0001] The invention relates to the technical field of biological control, and in particular to a solanacearum phage resistant to chemical pesticides and application thereof. Background Art

[0002] Plant bacterial wilt is a devastating soil-borne disease caused by Ralstonia solanacearum, which can infect more than 450 plant species from 54 families, including olive, mulberry, pepper, potato, peanut, sesame, sweet potato, fig, mulberry, eucalyptus, etc. In my country, more than 30 provinces including Guangdong, Fujian, Sichuan and Hunan have reported damage caused by Ralstonia solanacearum.

[0003] At present, the main methods for the prevention and control of bacterial wilt are to cultivate disease-resistant varieties, chemical prevention and control, biological prevention and control, etc., but there are few varieties of plants resistant to bacterial wilt, the breeding process is complicated, and resistance is easy to disappear. The large-scale use of pesticides will also bring about problems with the balance of the ecosystem and human health. With the development of people's concepts, higher requirements have been put forward for the prevention and control of bacterial wilt. On the basis of effective prevention and control of bacterial wilt, it is also necessary to meet the development requirements of modern agriculture. As a potential biological control resource for bacterial wilt, the phage of Ralstonia solanacearumphage has great advantages due to its high host specificity and green safety.

[0004] Chinese patent publication number CN117625555A discloses a solanacearum phage isolated from soil and its microsphere preparation process, which provides a phage source for industrial production of phages for the prevention and treatment of crop bacterial wilt. Chinese patent publication number CN112029789B discloses the use of phage trp574 gene in reducing the resistance of solanacearum to phage, which can obtain transgenic solanacearum strains with reduced phage resistance, thereby improving the use and prevention effect of phage.

[0005] Chinese patents with publication numbers CN117247908A, CN115851615A and CN116622649A disclose a bacteriophage with a high lysis rate, high control efficiency, or a wide control range for Ralstonia solanacearum, but the above patents do not study the tolerance of the bacteriophage to chemical pesticides and its specificity to Ralstonia solanacearum.

[0006] At present, there are still few studies on the use of bacteriophages to control bacterial wilt. Using bacteriophages to lyse pathogens to control bacterial wilt has broad application prospects. However, there is a shortage of bacteriophage products for bacterial wilt on the market.

[0007] It should be noted that the information disclosed in the above background technology section is only used to enhance the understanding of the background of the present disclosure, and therefore may include information that does not constitute the prior art known to ordinary technicians in the field. Summary of the invention

[0008] In view of this, the present invention proposes a chemical pesticide-resistant Ralstonia solanacearum phage and application thereof, which prevents and controls plant bacterial wilt by lysing Ralstonia solanacearum.

[0009] The technical solution of the present invention is achieved as follows: the present invention provides a chemical pesticide-resistant Ralstonia solanacearumphage, the Ralstonia solanacearumphage is named Ralstonia solanacearumphage RSP35, and the Ralstonia solanacearum phage is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC M 2024498.

[0010] The present invention also provides the use of the above-mentioned chemical pesticide-resistant Ralstonia solanacearum phage in the preparation of a pharmaceutical preparation for preventing and / or treating bacterial wilt.

[0011] The present invention also provides the use of the above-mentioned chemical pesticide-resistant Ralstonia solanacearum phage in the preparation of a pharmaceutical composition for preventing and / or treating bacterial wilt.

[0012] The present invention also provides a pharmaceutical preparation, which comprises the above-mentioned Ralstonia solanacearum bacteriophage and a microbial pesticide acceptable carrier.

[0013] Based on the above technical solution, preferably, the pH of the pharmaceutical preparation is 3-10.

[0014] Based on the above technical solution, preferably, the storage temperature of the pharmaceutical preparation is 4-37°C.

[0015] The present invention also provides a pharmaceutical composition, which comprises a solanacearum phage and a chemical pesticide;

[0016] The solanacearum phage is the solanacearum phage RSP35;

[0017] The chemical pesticides include one or more chemical pesticides used for preventing or treating bacterial wilt.

[0018] Based on the above technical solution, preferably, the pH of the pharmaceutical composition is 3-10.

[0019] Based on the above technical solution, preferably, the storage temperature of the pharmaceutical composition is 4-37°C.

[0020] The solanacearum phage provided by the present invention has the following beneficial effects compared with the prior art:

[0021] 1. Ralstonia solanacearum phage RSP35 has strong specificity, has no destructive effect on other bacterial flora in the soil, and will not break the microbial balance in the soil. Compared with antibiotics and chemical antimicrobial agents, it has the characteristics of high specificity, no residue and high safety.

[0022] 2. R. solanacearum phage RSP35 has a high tolerance to a variety of chemical pesticides used to prevent and / or treat bacterial wilt. In other words, chemical pesticides do not affect the stability and potency of R. solanacearum phage RSP35.

[0023] 3. In terms of application, on the one hand, the preparations prepared by RSP35, a bacteriophage of Ralstonia solanacearum, can be used to antagonize Ralstonia solanacearum and can be used for the prevention, control and treatment of plant bacterial wilt. On the other hand, based on the high tolerance of RSP35, a bacteriophage of Ralstonia solanacearum to chemical pesticides, RSP35 can be prepared into a pharmaceutical composition with chemical pesticides for use, which increases the synergistic cooperation between the two in resisting Ralstonia solanacearum, thereby improving the control effect of the pharmaceutical composition on bacterial wilt.

[0024] Storage Instructions:

[0025] Ralstonia solanacearumphage RSP35 was sent to China Center for Type Culture Collection on March 14, 2024, with the deposit number CCTCC M 2024498. Address: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 This is a schematic diagram of the plaque of Ralstonia solanacearum phage RSP35 in one embodiment of the present disclosure.

[0028] Figure 2 This is a schematic diagram of the titer results of Ralstonia solanacearum phage RSP35 at different infection multiplicities in one embodiment of the present disclosure.

[0029] Figure 3 This is a schematic diagram of the titer results of Ralstonia solanacearum bacteriophage RSP35 after being stored at different temperatures for different periods of time in one embodiment of the present disclosure.

[0030] Figure 4This is a schematic diagram of the titer results of Ralstonia solanacearum phage RSP35 at different pH values ​​in one embodiment of the present disclosure. DETAILED DESCRIPTION

[0031] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] The term "chemical pesticide" refers to pesticides containing organic compounds or inorganic compounds, such as inorganic pesticides, organic synthetic pesticides, and natural organic pesticides. Chemical drugs in this application are defined as chemical pesticides used to prevent or treat bacterial wilt.

[0033] The term "microbial pesticide" refers to a biopesticide that uses bacteria, fungi, viruses, protozoa or genetically modified living microorganisms as active ingredients to control diseases, insects, weeds, rodents and other harmful organisms. The microbial drug in this application is defined as a pharmaceutical preparation prepared using Ralstonia solanacearumphage RSP35 for the prevention or treatment of bacterial wilt.

[0034] The term "microbial pesticide-acceptable carrier" relates to a pesticide-acceptable, non-toxic carrier or diluent, etc., which is defined in the present disclosure as a carrier used to formulate a biologically active anti-Ralstonia solanacearum phage preparation.

[0035] The term "TSB medium" is tryptone soy medium, wherein T is tryptone, S is soy, and B is broth.

[0036] The embodiments of the present disclosure provide a chemical pesticide-resistant Ralstonia solanacearumphage, which is named Ralstonia solanacearumphage RSP35. The Ralstonia solanacearumphage is currently deposited in the China Center for Type Culture Collection with a deposit number of CCTCC M 2024498 and a deposit date of March 15, 2024.

[0037] The Ralstonia solanacearum phage RSP35 has strong lytic activity against host cells and has good temperature stability below 37°C, especially at 4-37°C, and can be stored for a long time. In this way, the drugability of the Ralstonia solanacearum phage RSP35 is increased, and the Ralstonia solanacearum phage RSP35 can be prepared into a pesticide preparation for anti-Ralstonia solanacearum treatment of plants. The Ralstonia solanacearum phage RSP35 has a stable titer between pH 3-10 and is easy to store. When the concentration of the Ralstonia solanacearum phage RSP35 is 104 pfu / mL, the host bacteria concentration is 10 7 cfu / mL, and when the MOI was 0.001, the fermentation titer of R. solanacearum phage RSP35 was the highest, which was 2.2×10 10 pfu / mL.

[0038] Embodiments of the present disclosure also provide use of the above-mentioned chemical pesticide-resistant Ralstonia solanacearum phage in the preparation of a pharmaceutical preparation for preventing and / or treating bacterial wilt disease.

[0039] The embodiments of the present disclosure also provide the use of the above-mentioned chemical pesticide-resistant Ralstonia solanacearum phage in the preparation of a pharmaceutical composition for preventing and / or treating bacterial wilt disease.

[0040] The embodiments of the present disclosure also provide a pharmaceutical preparation, including the above-mentioned Ralstonia solanacearum phage and a microbial pesticide acceptable carrier. That is, the Ralstonia solanacearum phage and a suitable carrier are prepared into a pharmaceutical preparation for agricultural prevention and / or treatment of Ralstonia solanacearum. The carrier in the present disclosure can be produced or purchased according to methods well known to those skilled in the art. The pharmaceutical preparation can be in the form of a solution, a dispersant, a suspension, a granule, etc.; the method of use can be spraying, broadcasting, furrow application, or irrigation with water.

[0041] In one embodiment of the present disclosure, the pH of the pharmaceutical preparation is 3 to 10. Within this pH range, the R. solanacearum phage RSP35 in the pharmaceutical preparation can be stably present, with a stable titer and high lytic activity against host cells. For example, the pH of the pharmaceutical preparation can be pH=3, pH=4, pH=5, pH=6, pH=7, pH=8, pH=9 or pH=10.

[0042] In one embodiment of the present disclosure, the pharmaceutical preparation is stored at a temperature of 4-37° C. The pharmaceutical preparation can be stored at a temperature below 37° C., preferably between 4-37° C. Within this temperature range, the titer of the Ralstonia solanacearum phage RSP35 in the pharmaceutical preparation is stable, the storage time of the pharmaceutical preparation can be extended, and the pressure on the production line, transportation line and warehousing can be reduced.

[0043] The embodiments of the present disclosure also provide a pharmaceutical composition, which includes a solanacearum phage and a chemical pesticide. The solanacearum phage is the above-mentioned solanacearum phage RSP35, and the chemical pesticide includes one or more chemical pesticides used to prevent or treat bacterial wilt. The solanacearum phage RSP35 provided in the embodiments of the present disclosure has high tolerance to chemical pesticides against solanacearum, and the solanacearum phage RSP35 can be prepared into a pharmaceutical composition with at least one chemical pesticide. The synergistic anti-solanacearum effect of the solanacearum phage RSP35 and the chemical pesticide can improve the prevention and treatment effects of bacterial wilt.

[0044] Exemplarily, the Ralstonia solanacearum phage RSP35 can be mixed with at least one chemical pesticide against bacterial wilt to obtain a first class pharmaceutical composition. Furthermore, the pH of the first class pharmaceutical composition is adjusted to 3-10 to improve the titer stability and host cell lysis activity of the Ralstonia solanacearum phage RSP35 in the first class pharmaceutical composition. For example, the pH of the first class pharmaceutical composition can be pH=3, pH=4, pH=5, pH=6, pH=7, pH=8, pH=9 or pH=10. Preferably, the first class pharmaceutical composition is stored at 4-37°C, so that the first class pharmaceutical composition has a stable titer and a prolonged shelf life.

[0045] Exemplarily, the Ralstonia solanacearum phage RSP35 can also be combined with at least one chemical pesticide against bacterial wilt to form a combination product. That is to say, in this product, the Ralstonia solanacearum phage RSP35 and the chemical pesticide are not mixed and are used at the same time. The combination product is defined as a second-class pharmaceutical composition. In the second-class pharmaceutical composition, the Ralstonia solanacearum phage RSP35 is stored at a pH of 3 to 10 and a temperature of 4-37°C; and this requirement is not required for the pH of the chemical pesticide. In this way, the second-class pharmaceutical composition is suitable for the preparation of a combination product for use against Ralstonia solanacearum with chemical pesticides that are unstable at a pH of 3 to 10 and the Ralstonia solanacearum phage RSP35.

[0046] The Ralstonia solanacearum phage RSP35 and its application provided by the present disclosure are further described below in conjunction with specific examples.

[0047] Example 1: Isolation and purification of Ralstonia solanacearum bacteriophage RSP35

[0048] The soil sample from which Ralstonia solanacearum phage RSP35 was isolated was taken from a tobacco planting base in Hunan Province.

[0049] (1) 10 g of tobacco rhizosphere soil was placed in 90 mL of sterile water and mixed, and cultured in a shaker at 30° C. and 180 rpm for 1 h. The cultured soil aqueous solution was centrifuged at 8000 rpm for 10 min to obtain a first supernatant, and the first supernatant was filtered with a 0.22 μm filter to obtain a filtrate.

[0050] (2) Take 2 mL of the filtrate, add 2 mL of 2-fold diluted TSB liquid medium, 400 μL of Ralstonia solanacearum culture medium (10 8 cfu / mL) were mixed evenly, and then enriched and cultured at 30°C, 200 rpm for 17 h.

[0051] (3) The culture medium after the shaking enrichment culture was centrifuged at 6000 rpm for 10 min to obtain a second supernatant, and the second supernatant was filtered and sterilized using a 0.22 μm sterile filter to obtain a phage stock solution.

[0052] (4) Take the host bacterial solution (10 8 cfu / mL) was mixed evenly with TSB semi-solid medium and poured onto a nutrient agar plate. After solidification, 5 μL of the phage stock solution obtained in step (3) was dripped on the plate. After drying, the plate was placed in an incubator at 30°C and inverted for 17-24 hours to obtain multiple plaque A.

[0053] (5) Select phages with obvious plaque A, dilute the phage enrichment solution 10 times with sterile water to obtain dilution solution, take 1 mL of the dilution solution and mix it with 400 μL of host bacterial solution (10 8 cfu / mL) and mixed evenly. After standing for 15 min, add about 7 mL of TSB semi-solid medium, pour it on the bottom plate of nutrient agar and mix evenly. After the semi-solid solidifies, place it in an incubator at 30°C and culture it upside down for 17-24 h to observe the growth of plaque B.

[0054] (6) On the double-layer plate where plaque B was formed, a large and transparent single plaque B was picked up with a sterile pipette tip, inoculated into 3 mL of TSB liquid culture medium, and 200 μL of Ralstonia solanacearum liquid was added and mixed. After overnight culture at 200 rpm at 30°C, the supernatant was centrifuged at 6000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm microporous filter membrane to obtain a filtrate. The filtrate was cultured using the double-layer plate method to obtain plaque C, and the morphology of plaque C was observed.

[0055] (7) Repeat step (6) for 3-5 times on plaque C to obtain target plaques of uniform shape and size.

[0056] (8) The target plaque was isolated to obtain R. solanacearum phage RSP35, such as Figure 1 As shown, the phage produced a single circular plaque with a diameter of about 2 mm on the lawn of Ralstonia solanacearum after 24 hours of culture. After 48 hours of culture, the plaque increased significantly.

[0057] The Ralstonia solanacearumphage RSP35 was sent to the China Center for Type Culture Collection for preservation in 2024, and the classification name is: Ralstonia solanacearumphage RSP35, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, Wuhan University, preservation number: CCTCC M 2024498.

[0058] Example 2: Determination of the Optimal Fermentation Conditions of Ralstonia solanacearum Phage RSP35

[0059] Pick a single colony of the host bacteria and culture it in 2 mL TSB medium at 30°C and 220 rpm, until the OD600 of the host bacteria solution is above 1.6. Transfer it to 20 mL TSB culture medium at a ratio of 2% (v / v), and culture it at 30°C and 220 rpm until the early logarithmic period to obtain a host bacteria suspension.

[0060] The host bacterial suspension and R. solanacearum phage RSP35 were counted, and the same volume of R. solanacearum phage RSP35 with different concentrations was added to the host bacterial suspension and mixed evenly, and then the mixture was allowed to stand for 15 minutes, and then fermented in a shaker at 30°C and 200 rpm for 17 hours, and the fermentation liquid was centrifuged at 6000 rpm for 10 minutes, and the supernatant was filtered through a 0.22 μm microporous membrane and counted. The experiment was repeated 3 times.

[0061] The results are as follows Figure 2 As shown, when the concentration of RSP35 of Ralstonia solanacearum was 10 4 pfu / mL, the host bacteria concentration is 10 7 cfu / mL, and when the MOI was 0.001, the fermentation titer of R. solanacearum phage RSP35 was the highest, which was 2.2×10 10 pfu / mL.

[0062] Example 3: Experiment on the lysis range of Ralstonia solanacearum by RSP35 bacteriophage

[0063] This example uses the spot method to determine the lysis effect of Ralstonia solanacearum bacteriophage RSP35 on different strains. 50 different strains were prepared in advance, and the specific strains and strain numbers are shown in Table 1.

[0064] Each strain was inoculated into a centrifuge tube containing 2 mL of TSB medium and cultured at 30°C and 220 rpm for 24 h to obtain a bacterial suspension of each strain (10 8 cfu / mL). 400 μL of bacterial suspension was mixed with TSB semi-solid medium and spread on the prepared nutrient agar plate. Ralstonia solanacearum phage RSP35 (10 9 pfu / mL) was diluted 1000 times, and 5 μL of the diluted Ralstonia solanacearum phage RSP35 was dripped on the above nutrient agar. After drying, it was cultured at 30°C for 24 hours and the results were observed. The experiment was repeated three times, and the statistical results are shown in Table 1.

[0065] Table 1. Lysis effect of RSP35 of Ralstonia solanacearum on different strains

[0066] strain Number of strains Lysis rate% Ralstonia solanacearum Ⅰ(15) 19 88.9％ Ralstonia solanacearum Ⅰ(34) 6 66.7％ Ralstonia solanacearum Ⅰ(17) 2 88.2％ Ralstonia solanacearum Ⅱ 3 80％ Bacillus subtilis 8 0 Actinomycetes 5 0 Pseudomonas 7 0

[0067] Example 4: Tolerance of Ralstonia solanacearum phage RSP35 to different pesticides

[0068] Ralstonia solanacearum phage RSP35 was mixed with fungicides (anti-Ralstonia solanacearum chemical pesticides) of different dilutions, and the final concentration of Ralstonia solanacearum phage RSP35 was 10 9 pfu / mL, the final concentration of each fungicide was determined according to the dilution multiple in Table 2, and the mixture of R. solanacearum phage RSP35 and sterile water was used as the control group. For example, referring to Table 2, the mixture of R. solanacearum phage RSP35 and 3% Zhongshengmycin wettable powder was as follows: sterile water was added to 3% Zhongshengmycin wettable powder at a mass volume ratio of 1:600 ​​to obtain a diluted Zhongshengmycin solution, and R. solanacearum phage RSP35 was added to the Zhongshengmycin solution, and the final concentration of R. solanacearum phage RSP35 was 10 9 The mixing method of R. solanacearum phage RSP35 and 20% thiamethoxam suspension was as follows: sterile water was added to 20% thiamethoxam suspension at a mass volume ratio of 1:300 to obtain a diluted thiamethoxam suspension, and R. solanacearum phage RSP35 was added to the thiamethoxam suspension. The final concentration of R. solanacearum phage RSP35 was 10 9 The mixing method of Ralstonia solanacearum phage RSP35 and 46% copper hydroxide water dispersible granules is as follows: sterile water is added to 46% copper hydroxide water dispersible granules at a mass volume ratio of 1:1000 to obtain a diluted copper hydroxide solution, and Ralstonia solanacearum phage RSP35 is added to the copper hydroxide solution. The final concentration of Ralstonia solanacearum phage RSP35 is 10 9 The mixing method of R. solanacearum phage RSP35 and 50% chlorobromoisocyanuric acid soluble powder is as follows: sterile water is added to 50% chlorobromoisocyanuric acid soluble powder at a mass volume ratio of 1:1000 to obtain a diluted chlorobromoisocyanuric acid solution, and R. solanacearum phage RSP35 is added to the chlorobromoisocyanuric acid solution. The final concentration of R. solanacearum phage RSP35 is 10 9 pfu / mL.

[0069] The mixed solution of each fungicide in Table 1 and R. solanacearum phage RSP35 was stored in a 30°C incubator, and the mixed solution corresponding to each fungicide was repeated 3 times. Samples were taken every 2 days (d) to determine the R. solanacearum phage RSP35 titer (pfu / mL), and the determination was continued for 10 days. The R. solanacearum phage RSP35 titer (pfu / mL) is the number of R. solanacearum phage RSP35 formed on the plate per milliliter of R. solanacearum phage RSP35 solution.

[0070] According to Table 2, compared with the control group, the titer (pfu / mL) of R. solanacearum phage RSP35 after mixing with various antimicrobial agents on the 2nd day (2d), 4th day (4d) and 6th day (6d) was basically equivalent to that of the control group. On the 8th day (8d) and the 10th day (10d), except for the reduction of the titer of RSP35 in the mixed solution of copper hydroxide solution and RSP35, the titer of RSP35 in the other groups after mixing with antimicrobial agents was not affected, and the titer was basically equivalent to that of the control group. This shows that RSP35 has good tolerance to common chemical pesticides for bacterial wilt, including 3% zhongshengmycin wettable powder, 20% thiamethoxam suspension, 20% kasugamycin water dispersible granules, 42% trichloroisocyanuric acid wettable powder, and 50% chlorobromoisocyanuric acid.

[0071] Table 2. Determination of the potency of R. solanacearum phage RSP35 after mixing with different fungicides

[0072]

[0073]

[0074] Example 5: Temperature stability test of Ralstonia solanacearum bacteriophage RSP35

[0075] Take 6 portions of 9 mL TSB liquid culture medium into 15 mL sterile centrifuge tubes and place them in constant temperature environments at 4°C, 25°C, 37°C, 50°C, 60°C, and 70°C, respectively. After the temperature is balanced, add 1 mL of 2.2×10 10 pfu / mL pure culture of R. solanacearum phage RSP35. Cultivate in a constant temperature environment at the corresponding temperature, and take R. solanacearum phage RSP35 from each tube at each temperature for titer determination at 1h, 2h, 6h, and 24h. Take another 6 15mL sterile centrifuge tubes containing 9mL TSB liquid culture medium, culture in duplicate and take the average value, and repeat the experiment 3 times.

[0076] The results are as follows Figure 3 As shown, in TSB liquid culture medium, the activity of R. solanacearum phage RSP35 changes little at temperatures below 45°C, has good temperature stability at 4-37°C, and can be stored for a long time.

[0077] Example 6, pH stability test of Ralstonia solanacearum bacteriophage RSP35

[0078] Take 11 portions of sterile water, adjust the pH values ​​to pH 2–pH 12 (pH integer values), and filter with a 0.22 μm microporous membrane. Take 9 mL of sterile water with different pH values ​​and add 1 mL of R. solanacearum bacteriophage RSP35 fermentation broth (2.2×10 10pfu / mL), mixed evenly, and allowed to stand at 25°C for 2 h before determining the titer of Ralstonia solanacearum phage RSP35.

[0079] The results are as follows Figure 4 As shown, the titer of R. solanacearum phage RSP35 changes little under the condition of pH 3-10, and has good pH stability.

[0080] Example 7: Potted plant test of Ralstonia solanacearum phage RSP35

[0081] Potted test: 108 tobacco seedlings with healthy appearance and 3 true leaves were selected and moved into pots. They were randomly divided into 6 treatment groups, namely RSP35 prevention group, RSP35 treatment group, thiamethoxam group, infection group, compound group and blank group. Each treatment had 3 replicates and 6 tobacco seedlings in each replicate. The experiment started when the true leaves of the tobacco seedlings grew to 5 leaves. The results were investigated and recorded one month after the pathogen was inoculated. Among them, the thiamethoxam suspension used in the thiamethoxam group and the compound group was: sterile water was added to 20% thiamethoxam suspension at a volume ratio of 1:300 to prepare the thiamethoxam suspension.

[0082] (1) RSP35 prevention group: 50 mL 5.0×10 9 pfu / mL RSP35 solution of Ralstonia solanacearum phage. After 24 h, a sterilized blade was used to cut one cut on each side of the main root of the tobacco seedlings and watered with the Ralstonia solanacearum suspension (1.0×10 8 cfu / mL)20mL / strain.

[0083] (2) RSP35 treatment group: A sterilized blade was used to cut one cut on each side of the taproot of the tobacco seedlings, and the seedlings were irrigated with a suspension of Ralstonia solanacearum (1.0×10 8 cfu / mL) 20mL / strain, 24h later, 5.0×10 9 pfu / mL of Ralstonia solanacearum phage RSP35, 50mL / plant per root.

[0084] (3) Thiam copper group: Use a sterilized blade to make a cut on both sides of the main root of the tobacco seedlings, and water them with a suspension of Ralstonia solanacearum (1.0×10 8 cfu / mL) 20mL / plant, and 24h later, 50mL / plant of thiamethoxam suspension was used for irrigation.

[0085] (4) Combined group: Use a sterilized blade to make a cut on both sides of the main root of the tobacco seedlings, and water them with a suspension of Ralstonia solanacearum (1.0×10 8 cfu / mL) 20mL / plant, 24h later, 25mL / plant of thiamethoxam suspension was irrigated, and the concentration of irrigated was 5.0×10 9 pfu / mL of Ralstonia solanacearum phage RSP35, 25mL / plant per root.

[0086] (5) Infection group: Use a sterilized blade to make a cut on both sides of the main root of the tobacco seedlings, and water them with a suspension of tobacco solanacearum (1.0×10 8 cfu / mL) 20mL / plant; 24h later, irrigate with 50mL / plant of sterile water.

[0087] (6) Blank group: irrigate with 20 mL of sterile water per plant. After 24 hours, make a cut on each side of the main root of the tobacco seedlings and irrigate with 50 mL of sterile water per plant.

[0088] Disease classification standard (in units):

[0089] Level 0: The whole plant is disease-free;

[0090] Level 1: There are occasional chlorotic spots on the stem, or less than 1 / 2 of the leaves on the diseased side wilt;

[0091] Level 3: There are black streaks on the stem, but not exceeding 1 / 2 of the stem height, or 1 / 2 to 2 / 3 of the leaves on the diseased side wilt;

[0092] Level 5: The black streaks on the stem exceed 1 / 2 of the stem height but do not reach the top of the stem, or more than 2 / 3 of the leaves on the diseased side wilt;

[0093] Level 7: Black streaks on the stem reach the top of the stem, or all leaves of the diseased plant wilt;

[0094] Level 9: The diseased plants are basically dead.

[0095] Disease index = Σ(number of diseased plants × representative value of the disease level) / (total number of plants surveyed × highest representative value) × 100

[0096] Control effect = (control disease index - treatment disease index) / control disease index × 100%.

[0097] Table 3. Control effect of each treatment group on tobacco bacterial wilt

[0098] Treatment Group Incidence Disease index Relative efficacy RSP35 prevention group 61.1％ 50.2％ 32.61％ RSP35 treatment group 56.5％ 48.2％ 38.60％ Thiessen copper group 57.4％ 50.1％ 36.2％ Composite Group 46.3％ 33.9％ 56.8％ Infection group 93.1％ 78.5％ ～ Blank Group 0％ 0％ ～

[0099] As shown in Table 3, the RSP35 prevention group, RSP35 treatment group, copper-thiamethoxam group and compound group can effectively reduce the incidence and disease index of tobacco bacterial wilt and control the further infection of RSP35. At the same time, the combination of RSP35 and copper-thiamethoxam has a synergistic effect. This experiment shows that bacteriophages have the potential to be developed into green and environmentally friendly biological agents, and can also be used to prepare compound products with other anti-RSP35 drugs.

[0100] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A chemical pesticide-resistant Ralstonia solanacearum phage, characterized in that: The solanacearum phage is named as solanacearum phage RSP35, and the solanacearum phage is preserved in China Center for Type Culture Collection with a preservation number of CCTCC M 2024498.

2. Use of the chemical pesticide-resistant Ralstonia solanacearum phage according to claim 1 in the preparation of a pharmaceutical preparation for preventing and / or treating bacterial wilt.

3. Use of the chemical pesticide-resistant Ralstonia solanacearum phage according to claim 1 in the preparation of a pharmaceutical composition for preventing and / or treating bacterial wilt.

4. A pharmaceutical preparation, characterized in that It comprises the solanacearum phage described in claim 1 and a microbial pesticide acceptable carrier.

5. The pharmaceutical preparation according to claim 4, characterized in that The pH of the pharmaceutical preparation is 3-10.

6. The pharmaceutical preparation according to claim 4, characterized in that The storage temperature of the pharmaceutical preparation is 4-37°C.

7. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises a solanacearum phage and a chemical pesticide; The Ralstonia solanacearum phage is the Ralstonia solanacearum phage according to claim 1; The chemical pesticides include one or more chemical pesticides used for preventing or treating bacterial wilt.

8. The pharmaceutical composition according to claim 7, characterized in that The pH of the pharmaceutical composition is 3-10.

9. The pharmaceutical composition according to claim 7, characterized in that The storage temperature of the pharmaceutical composition is 4-37°C.

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