Ralstonia solanacearum bacteriophage YL1 and application thereof

Abstract

The invention discloses a ralstonia solanacearum bacteriophage YL1 and application thereof. Belongs to the technical field of biological control. The bacteriophage can specifically split ralstonia solanacearum, and the bacteriophage has the highest activity, the strongest splitting capacity and the wide splitting spectrum at the temperature of 30-60 DEG C and the pH value of 4-11. An indoor test shows that the cracking rate of the strain on 23 bacterial wilt bacteria reaches 86.96%, a greenhouse pot experiment shows that the control effect of the strain is 90.82 + / -2.20%, and the strain has a good biocontrol effect on tobacco bacterial wilt.

Description

Technical Field

[0001] The invention relates to the technical field of biological control, and more specifically to a tobacco solanacearum phage YL1 and application thereof. Background Art

[0002] Tobacco bacterial wilt is the number one bacterial disease in tobacco production and is a typical soil-borne disease. Pathogens that overwinter and summer in the soil infect tobacco roots and colonize in the xylem vessels. The pathogens multiply in large numbers and secrete extracellular polysaccharides to block the vessels, resulting in obstruction of water and nutrient transport. The aboveground part wilts and dies because the water consumed by transpiration cannot be replenished in time. The prevention and control of tobacco bacterial wilt is extremely difficult. Conventional chemical pesticides have poor control effects on it, and there is a lack of high-quality wilt-resistant varieties, so the disease is serious. Exploring biological control resources and developing new biocontrol microbial preparations to break down the bacterial wilt bacteria in the soil and quickly reduce the population of the bacterial wilt bacteria in the soil can greatly reduce the harm of bacterial wilt, which is one of the research hotspots in the field of biological control of tobacco bacterial wilt.

[0003] Bacteriophages are viruses that specifically infect bacteria and are ubiquitous in the environment. Bacteriophages have the characteristics of fast infection speed, short lysis time and high specificity for hosts. They can reduce the number of host bacteria in the environment in a short period of time without negative effects on non-host organisms. They have good safety and application prospects. The treatment method of screening, purifying and enriching specific virulent bacteriophages for specific pathogenic bacteria and releasing them into specific ecological environments such as soil, water or animal intestines to control pathogenic bacteria is called "phage therapy". Since the discovery of bacteriophages, phage therapy has been widely used in the prevention and control of human, animal and plant diseases, especially in the prevention and control of multi-drug resistant pathogenic bacteria. It has shown unique advantages and is considered to be one of the most promising technologies for the prevention and control of bacterial diseases.

[0004] In summary, how to provide a tobacco solanacearum phage is an urgent problem that needs to be solved by those skilled in the art. Summary of the invention

[0005] In view of this, the present invention provides a tobacco bacterial wilt bacteriophage YL1 and its application. By analyzing the biological characteristics of the phage and conducting pot experiments to explore its control effect on tobacco bacterial wilt, it is found that the application of phage YL1 can effectively prevent and control tobacco bacterial wilt, and phage YL1 has strong host specificity, does not kill other bacteria in the soil, and has no negative effect on the soil microecological environment.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A tobacco solanacearum phage YL1, named Ralstonia Phage YL1, was classified as Ralstonia Phage and was deposited in the China Center for Type Culture Collection on October 23, 2024, with the deposit number CCTCCNO: M 20242304. The deposit address is Wuhan University, Wuhan, China.

[0008] Furthermore, the nucleic acid type of the bacteriophage YL1 is double-stranded, belonging to the phylum Caudoviricetes, the class Caudoviricetes, the order Caudovirales, and the genus Gervaiseviru.

[0009] Furthermore, the titer of the bacteriophage YL1 is ≥10 10 PFU / mL.

[0010] Furthermore, the suitable survival temperature of the bacteriophage YL1 is 30-60°C, the suitable pH is 4-11, and the lethal temperature is 80°C.

[0011] The application of the above-mentioned bacteriophage YL1 in lysing Ralstonia solanacearum.

[0012] Furthermore, the solanacearum is solanacearum GMI1000.

[0013] The application of the above-mentioned bacteriophage YL1 in preventing and controlling plant wilt disease.

[0014] Furthermore, the plant is tobacco.

[0015] A preparation for preventing and treating bacterial wilt, comprising the above-mentioned bacteriophage YL1.

[0016] Furthermore, the content of bacteriophage YL1 was ≥1×10 8 PFU / mL.

[0017] A method for preventing and controlling tobacco bacterial wilt comprises watering the above preparation to the roots of tobacco plants.

[0018] Furthermore, the preparation should be stored away from light and at low temperature (4° C.) for future use.

[0019] It can be seen from the above technical solution that, compared with the prior art, the beneficial effects achieved by the present invention are:

[0020] The bacteriophage YL1 of the present invention can specifically lyse tobacco bacterial wilt pathogens, and the bacteriophage activity is the highest in the temperature range of 30-60°C and pH=4-11, the lysing ability is the strongest, and the lysing spectrum is wide. Indoor tests show that the lysing rate of 23 strains of bacterial wilt pathogens is 86.96%, and greenhouse pot tests show that the control effect is 90.82±2.20%, and the bacteriophage has a good biological control effect on tobacco bacterial wilt pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] 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 embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0022] Figure 1 The plaques formed by bacteriophage YL1 on a double-layer plate in Example 2 of the present invention;

[0023] Figure 2 The appearance of bacteriophage YL1 in Example 3 of the present invention under an electron microscope;

[0024] Figure 3 is the phylogenetic tree of bacteriophage YL1 in Example 5 of the present invention;

[0025] Figure 4 is the optimal multiplicity of infection of bacteriophage YL1 in Example 7 of the present invention;

[0026] Figure 5 is the temperature tolerance of bacteriophage YL1 in Example 9 of the present invention;

[0027] Figure 6 is the pH tolerance of bacteriophage YL1 in Example 10 of the present invention;

[0028] Figure 7 This is a picture of a potted plant experiment of bacteriophage YL1 in Example 11 of the present invention for preventing and controlling tobacco bacterial wilt. DETAILED DESCRIPTION

[0029] The following will be combined with the drawings in 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.

[0030] The drugs required for the present invention are conventional experimental drugs, which are purchased from commercial channels; the experimental methods not mentioned are conventional experimental methods and will not be described in detail here.

[0031] Example 1

[0032] Isolation, identification and cultivation of Ralstonia solanacearum

[0033] Use an inoculation loop to pick up a small amount of bacteria from the pus overflowing from the diseased tissue of tobacco bacterial wilt diseased plants, streak on LB medium, culture at 37℃ for 48h, pick colonies, amplify 16S rRNA of the test strain with bacterial universal primer 1492R / 27F, PCR amplification (program is 95℃5min; 95℃30s, 59℃30s, 72℃90s, 34cycles; 72℃10min), detect and recover PCR amplification products (about 1500bp) by 1.2% agarose gel electrophoresis, and send to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. Comparison of DNA sequence with NCBI database shows that the test strain is Ralstoniapseudosolanacearum. Inoculate a single colony of the strain into LB liquid medium, culture at 30℃, 180r / m shaking overnight, and obtain bacterial solution of Ralstonia pseudosolanacearum.

[0034] The resulting 16S rRNA sequence is:

[0035]

[0036] Example 2

[0037] Isolation and purification of bacteriophage

[0038] 5 ml of sterile water was added to 2 g of rhizosphere soil sample taken from tobacco fields in Huayuan County, Xiangxi Autonomous Prefecture, and the mixture was vortexed and homogenized. The mixture was allowed to stand for 30 min, and then centrifuged at 13000 r / m for 8 min at room temperature. The supernatant was collected and sterilized by filtration with a 0.22 μm aqueous filter. The filtrate and the solanacearum solution provided in Example 1 were mixed in equal proportions, added to a triangular flask containing 40 mL of LB liquid culture medium, and cultured overnight at 30°C and 160 r / m. 10 mL of the co-culture solution was centrifuged, the supernatant was sterilized by filtration with a 0.22 μm aqueous filter, and gradiently diluted to 10 -2 ~10 -4 , and then 100 μL of host bacteria (the solanacearum solution prepared in Example 1) and 100 μL of the gradient dilution solution were taken, vortexed and mixed, and then spread on a double-layer plate, and cultured at 30°C for 12 hours. The plaque was picked and added to 1.6 mL of LB liquid culture medium, and then 40 μL of solanacearum solution was inoculated, and cultured at 30°C and 180 r / m until the turbid bacterial solution became clear and filamentous lysates could be observed. The bacterial solution was centrifuged at 13000 r / m for 7 minutes, and the supernatant was filtered through a 0.22 μm aqueous phase filter and gradiently diluted to 10 -8 ~10 -9 , get the phage dilution, then take 70 μL of the host bacteria and phage dilution, vortex mix, spread on double-layer plates, and culture at 30°C until plaques appear ( Figure 1 ), repeat the above operation 4 times, and the purified phage is named YL1.

[0039] Example 3

[0040] Observation of the appearance of bacteriophage YL1 using transmission electron microscopy

[0041] The solanacearum solution and bacteriophage YL1 prepared in Example 1 were inoculated at an inoculum rate of 0.3% (V / V), cultured overnight, and bacteriophage YL1 culture solution was prepared, which was then concentrated. 10 μL of the concentrated bacteriophage YL1 culture solution was taken and precipitated on a copper mesh for 1 min, and the floating liquid was removed by filter paper. Then 10 μL of phosphotungstic acid staining solution was dripped on the copper mesh to cover the phage liquid surface for 1 min. The excess liquid was removed by filter paper and then dried at room temperature for 10 min. The image was detected by electron microscopy (100-120 kV). The results of transmission electron microscopy showed that the head of bacteriophage YL1 was polyhedral, about 50 nm wide, and the tail was shorter, about 30 nm long. According to the classification standards of the International Committee on Taxonomy of Viruses (ICCV), it was identified as Brachyuraviridae ( Figure 2 ).

[0042] Example 4

[0043] Extraction of nucleic acid and genome sequencing of bacteriophage YL1

[0044] First, phage YL1 and Ralstonia solanacearum liquid were inoculated into 50 mL LB liquid culture medium at an equal ratio of 0.5% (V / V), cultured overnight at 30°C and 180 rpm until filamentous lysates appeared, DNase I and RNase A were added to a final concentration of 0.8 μg / mL, and the culture was allowed to stand at 30°C for 30 min to digest the nucleic acid of Ralstonia solanacearum.

[0045] Secondly, sodium chloride was added to a final concentration of 1 mol / L, ice-bathed for 1 h, centrifuged at 13000 r / m for 10 min, PEG 8000 was added to the supernatant to a final concentration of 10%, ice-bathed for 3 h, centrifuged at 13000 r / m for 20 min, the supernatant was discarded, and the precipitate was resuspended in 2 mL SM buffer.

[0046] Third, take 600 μL of the resuspension, add 30 μL of proteinase K and 600 μL of CTAB, and place in a 65°C water bath for 10 min. Invert and mix three times during the process. Then add 700 μL of phenol: chloroform: isoamyl alcohol (25:24:1), vortex to mix, let stand for 10 min, centrifuge at 13000 r / m for 10 min, collect the upper aqueous phase and add an equal volume of chloroform: isoamyl alcohol (24:1) to extract to remove the protein.

[0047] Fourth, centrifuge at 13000 r / m for 10 min, collect the upper aqueous phase, add an equal volume of anhydrous ethanol, let stand at -20°C for 30 min, centrifuge at 13000 r / m for 10 min, discard the supernatant, and add 120 μL ddH2O to dissolve the phage nucleic acid after the ethanol evaporates.

[0048] Finally, the purity of the phage genomic DNA was detected by 1.5% agarose gel electrophoresis and sent to Wuhan Fraser Gene Information Co., Ltd. for sequencing.

[0049] Example 5

[0050] Construction of phylogenetic tree of bacteriophage YL1

[0051] The genome sequence of phage YL1 was compared with the existing phage sequences in the NCBI database, and a phylogenetic tree was constructed using MEGA 10.0 with Pseudomonas phage Itty13 as the outgroup. It was found that phage YL1 and RalstoniaPhage QkW1 were on the same branch, and thus it was identified as a short-tailed phage ( Figure 3 ).

[0052] Example 6

[0053] preservation

[0054] The bacteriophage provided by the present invention is named Ralstonia Phage YL1, and its classification name is RalstoniaPhage. It was deposited in the China Center for Type Culture Collection on October 23, 2024, with a deposit number of CCTCC NO: M20242304, and the deposit address is Wuhan University, Wuhan, China.

[0055] Example 7

[0056] Optimal multiplicity of infection (MOI) of bacteriophage YL1

[0057] The solanacearum solution and phage YL1 prepared in Example 1 were inoculated at an inoculum volume of 0.3% (V / V), and the concentration was determined by overnight culture. Then, according to the infection complex of MOI=3, 2, 1, 0, -1, -2, -3 (Table 1), they were incubated at 30°C and the optimal pH for 6h, and then the phage titers of different ratios were determined by the double-layer plate method. Then, the lysis curve was determined: equal proportions of phage culture solution and host were added to a 48-well plate, co-cultured at 30°C for 12h, and the OD of the co-culture solution from 0 to 12h was measured using an enzyme marker (TECAN spark). 600 The cleavage curve was drawn.

[0058] Table 1 Phage MOI ratio table

[0059]

[0060] The results of determining the MOI of phage YL1 showed that the optimal MOI of phage YL1 was 2 ( Figure 4 ), the lysis curve of phage YL1 against Ralstonia solanacearum was determined according to the optimal MOI ratio, and it was found that the OD of the co-culture solution within 4 hours after adding the phage 600 The OD of the control (Ralstonia solanacearum) 600 It continued to increase within 12 hours, reaching 0.429 at 3 hours and 1.128 at 12 hours. The experimental results showed that when MOI=2, the phage could produce more offspring.

[0061] Example 8

[0062] Determination of lysis spectrum of bacteriophage YL1

[0063] The 23 tested tobacco bacterial wilt strains were streaked and activated, and single colonies were inoculated into 10 mL LB liquid culture medium at 30°C and 180 r / m for overnight shaking. The overnight cultured bacterial wilt strains and the phage YL1 culture solution were inoculated into 10 mL LB at an equal ratio of 0.5% (V / V), and the phage YL1 culture solution was not added as the control. After co-culture at 30°C and 180 r / m for 6 hours, the OD of the culture solution of the treatment group and the control group was measured. 600 .

[0064] The lysis activity of bacteriophage YL1 against different Ralstonia solanacearum is shown in Table 2. 600 The ratio of the mean to CK defines the lysis efficiency (+: lysis efficiency ≤ 40%; ++: lysis efficiency 40-80%; +++: lysis efficiency ≥ 80%). The results showed that phage YL1 could lyse 86.96% of the tested Ralstonia solanacearum strains, indicating that phage YL1 has a wide lysis spectrum and high lysis activity.

[0065]

[0066] Table 2 Lysis spectrum of phage YL1 against different Ralstonia solanacearum (in OD 600 Value measurement)

[0067]

[0068]

[0069] Example 9 Temperature tolerance of bacteriophage YL1

[0070] Prepare the solanacearum solution and phage YL1 culture solution as described above. Place phage YL1 in a water bath at 30°C, 37°C, 50°C, 60°C, 70°C, and 80°C for 1 h, and repeat each temperature gradient 3 times. The titer ( Figure 5 ). The results showed that the optimum temperature was 30-60℃ and the lethal temperature was 80℃.

[0071] Example 10

[0072] Acid and alkaline tolerance of bacteriophage YL1

[0073] Prepare the culture medium of Ralstonia solanacearum and phage YL1 as described above. Adjust the pH of water to 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0 (990 μL) with NaOH or HCl, then add 10 μL of phage YL1, incubate at 30°C for 1 h, repeat 3 times for each pH, ​​and determine the titer by double-layer plate method ( Figure 6 The results showed that the activity of phage YL1 remained stable in the pH range of 4 to 11, indicating that phage YL1 has good tolerance to the above pH.

[0074] Embodiment 11

[0075] Methods of administering bacteriophage preparations

[0076] The pot experiment was set up with 2 treatments, 3 replicates per treatment, and 16 tobacco seedlings per replicate.600 Adjust to 0.1, and inoculate 10 mL of Ralstonia solanacearum solution per tobacco seedling. Prepare the bacteriophage YL1 culture solution according to the above method and dilute it to 1×10 8 PFU / ml, each tobacco seedling was inoculated with 50mL of phage YL1 culture solution (direct root irrigation). The greenhouse temperature was set at 30℃, and the light / dark period was 12h / 12h. 14 days after inoculation, the disease level of each tobacco seedling was investigated plant by plant according to the national standard GB / T 23222-2008, and the disease index and control effect were calculated. The results showed that phage YL1 had a good control effect on bacterial wilt, with a control efficiency of 90.82%, which was good (Table 3, Figure 7 ).

[0077] Table 3 Evaluation of potted plant protection effect of YL1

[0078]

[0079] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0080] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A tobacco solanacearum phage YL1, characterized in that: Named Ralstonia Phage YL1, its classification name is Ralstonia Phage, and it was deposited in the China Center for Type Culture Collection on October 23, 2024, with the deposit number CCTCCNO:M 20242304, and the deposit address is Wuhan University, Wuhan, China.

2. Use of the bacteriophage YL1 described in claim 1 in lysing Ralstonia solanacearum.

3. The use according to claim 2, characterized in that The solanacearum is solanacearum GMI1000.

4. Use of the bacteriophage YL1 according to claim 1 in preventing and controlling plant bacterial wilt.

5. The use according to claim 4, characterized in that The plant is tobacco.

6. A preparation for preventing and treating bacterial wilt, characterized in that: Comprising the bacteriophage YL1 described in claim 1.

7. The preparation according to claim 6, characterized in that The content of bacteriophage YL1 is ≥1×10 8 PFU / mL.

8. A method for preventing and treating tobacco bacterial wilt, characterized in that: Water the preparation according to claim 6 or 7 to the roots of tobacco plants.

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