Tobacco bacterial wilt bacteriophage YL1 and application thereof

Abstract

The application discloses a tobacco Pseudomonas syringae bacteriophage YL1 and application thereof, and belongs to the technical field of biological control. The bacteriophage can specifically lyse the tobacco Pseudomonas syringae. The bacteriophage has the highest activity and the strongest lysis ability in the range of 30-60 DEG C and pH = 4-11, and has a wide lysis spectrum. Indoor test shows that the lysis rate of the bacteriophage to 23 strains of Pseudomonas syringae reaches 86.96%, and greenhouse potting test shows that the control effect of the bacteriophage reaches 90.82+ / -2.20%, so the bacteriophage has a good biocontrol effect on the tobacco Pseudomonas syringae.

Description

Technical Field

[0001] This invention relates to the field of biological control technology, and more specifically to a strain of Ralstonia solanacearum bacteriophage YL1 and its applications. Background Technology

[0002] Bacterial wilt of tobacco is the leading bacterial disease in tobacco production, and it is a typical soil-borne disease. The pathogen, which overwinters and oversummers in the soil, infects the tobacco root system and colonizes the xylem vessels. The pathogen multiplies rapidly and secretes extracellular polysaccharides that clog the vessels, hindering water and nutrient transport. As a result, the above-ground parts wilt and die because the water lost through transpiration cannot be replenished in time. Controlling bacterial wilt of tobacco is extremely difficult; conventional chemical pesticides are ineffective, and there is a lack of high-quality resistant varieties, leading to severe outbreaks. Exploring biological control resources and developing novel biocontrol microbial agents to lyse the bacterial wilt pathogen in the soil and rapidly reduce its population can significantly mitigate the damage caused by bacterial wilt, making it a research hotspot 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 are characterized by rapid infection, short lysis time, and high host specificity. They can quickly reduce the number of host bacteria in the environment without negatively impacting non-host organisms, demonstrating good safety and promising application prospects. The treatment method of controlling pathogenic bacteria by screening, purifying, and enriching specific virulent bacteriophages, and releasing them into specific ecological environments such as soil, water bodies, or animal intestines, is called "phage therapy." Since the discovery of bacteriophages, phage therapy has been widely used in the control of human, animal, and plant diseases, especially demonstrating unique advantages in controlling multidrug-resistant pathogens, and is considered one of the most promising technologies for controlling bacterial diseases.

[0004] In summary, how to provide a tobacco wilt bacteriophage is a problem that urgently 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 phage YL1 and its application. By analyzing the biological characteristics of this phage and conducting pot experiments, its control effect on tobacco bacterial wilt was explored. It was found that the application of phage YL1 can effectively control tobacco bacterial wilt, and phage YL1 has strong host specificity, does not kill other bacteria in the soil, and has no negative effects on the soil micro-ecological environment.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A bacteriophage YL1 of Ralstonia spp., named Ralstonia Phage YL1 and taxonomically named Ralstonia Phage, was deposited on October 23, 2024, at the China Center for Type Culture Collection (CCTCCNO: M 20242304) at Wuhan University, Wuhan, China.

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

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

[0010] Furthermore, the optimal survival temperature for the bacteriophage YL1 is 30–60°C, the optimal pH is 4–11, and the lethal temperature is 80°C.

[0011] The application of the aforementioned bacteriophage YL1 in lysing Ralstonia solanacearum.

[0012] Furthermore, the Ralstonia solanacearum is Ralstonia solanacearum GMI1000.

[0013] The application of the aforementioned bacteriophage YL1 in the prevention and control of bacterial wilt in plants.

[0014] Furthermore, the plant in question is tobacco.

[0015] An agent for the prevention and treatment of bacterial wilt includes the aforementioned bacteriophage YL1.

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

[0017] A method for preventing and controlling tobacco bacterial wilt involves irrigating the roots of tobacco plants with the aforementioned preparation.

[0018] Furthermore, the preparation should be stored in the dark and at a low temperature (4°C) for later use.

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

[0020] The bacteriophage YL1 of this invention can specifically lyse *Ralstonia solanacearum*, the causal agent of tobacco bacterial wilt. Its activity is highest and its lytic ability is strongest within a temperature range of 30–60°C and a pH range of 4–11, exhibiting a broad lytic spectrum. Indoor experiments showed a lysis rate of 86.96% against 23 strains of *Ralstonia solanacearum*, while greenhouse pot experiments demonstrated a control efficacy of 90.82 ± 2.20%, indicating good biocontrol effects against tobacco bacterial wilt. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0022] Figure 1 This refers to the phage plaque formed by phage YL1 on a double-layered petri dish in Example 2 of the present invention;

[0023] Figure 2 The morphology of bacteriophage YL1 in Example 3 of this invention is shown under an electron microscope.

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

[0025] Figure 4 This represents the optimal multiplicity of infection for phage YL1 in Example 7 of the present invention;

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

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

[0028] Figure 7 This is a pot experiment image of the control of tobacco bacterial wilt by bacteriophage YL1 in Example 11 of the present invention. Detailed Implementation

[0029] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] The reagents required for this invention are conventional experimental reagents, purchased from commercially available 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 culture of Ralstonia solanacearum

[0033] A small amount of bacterial cells was collected from the ooze of bacteria from diseased tissue of tobacco plants infected with bacterial wilt using an inoculation loop. The cells were streaked onto LB agar and incubated at 37°C for 48 hours. Colonies were then picked, and the 16S rRNA of the tested strain was amplified using universal bacterial primers 1492R / 27F. PCR amplification was performed (programmed as follows: 95°C 5 min; 95°C 30 s, 59°C 30 s, 72°C 90 s, 34 cycles; 72°C 10 min). The PCR product (approximately 1500 bp) was detected and recovered by 1.2% agarose gel electrophoresis and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The DNA sequence was compared with the NCBI database, and the results showed that the tested strain was *Ralstonia pseudosolanacearum*. Single colonies of this strain were inoculated into LB liquid medium and incubated overnight at 30°C with shaking at 180 rpm to obtain *Ralstonia pseudosolanacearum* culture.

[0034] The obtained 16S rRNA sequence is as follows:

[0035]

[0036] Example 2

[0037] Isolation and purification of bacteriophages

[0038] Add 5 ml of sterile water to 2 g of rhizosphere soil sample taken from a tobacco field in Huayuan County, Xiangxi Tujia and Miao Autonomous Prefecture, vortex to homogenize, let stand for 30 min, then centrifuge at 13000 r / m at room temperature for 8 min. Collect the supernatant and filter it through a 0.22 μm aqueous phase filter for sterilization. Mix the filtrate with the Ralstonia solanacearum solution provided in Example 1 in equal proportion, add it to an Erlenmeyer flask containing 40 mL of LB liquid medium, and incubate overnight at 30 °C and 160 r / m. Centrifuge 10 mL of the co-culture medium, filter the supernatant through a 0.22 μm aqueous phase filter for sterilization, and serially dilute to 10⁻⁶. -2 ~10 -4 The serially diluted solution was obtained. Then, 100 μL of the host bacteria (Ralstonia solanacearum suspension prepared in Example 1) and 100 μL of the serially diluted solution were vortexed and mixed, then plated on a double-layer plate and incubated at 30°C for 12 h. Plasma was picked and added to 1.6 mL of LB liquid medium, followed by inoculation with 40 μL of Ralstonia solanacearum suspension. The culture was stopped at 30°C and 180 rpm with shaking until the turbid suspension became clear and filamentous lysis products could be observed. The suspension was centrifuged at 13000 rpm for 7 min, and the supernatant was filtered through a 0.22 μm aqueous filter and serially diluted to 10. -8 ~10 -9 The phage dilution was obtained, and then 70 μL each of the host bacteria and the phage dilution were taken, vortexed to mix, and spread into a double-layer plate. The plate was incubated at 30°C until phage plaques appeared. Figure 1 Repeat the above operation 4 times, and name the purified phage YL1.

[0039] Example 3

[0040] Morphage YL1 morphology observed using transmission electron microscopy

[0041] The *Ralstonia solanacearum* suspension and bacteriophage YL1 prepared in Example 1 were inoculated at a rate of 0.3% (V / V) and cultured overnight to prepare bacteriophage YL1 culture medium, which was then concentrated. 10 μL of the concentrated bacteriophage YL1 culture medium was placed on a copper grid for 1 min to settle. The floating liquid was absorbed with filter paper, and then 10 μL of phosphotungstic acid staining solution was added to the copper grid to cover the bacteriophage surface for 1 min. Excess liquid was absorbed with filter paper, and the medium was air-dried at room temperature for 10 min. Electron microscopy (100-120 kV) was then used for imaging. Transmission electron microscopy showed that the head of bacteriophage YL1 was polyhedral, approximately 50 nm wide, and the tail was relatively short, approximately 30 nm long. According to the International Committee on Taxonomy of Viruses (ICCV), it was identified as belonging to the family Bacteriophageidae (Brachyphaeidae). Figure 2 ).

[0042] Example 4

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

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

[0045] Next, add sodium chloride to a final concentration of 1 mol / L, incubate on ice for 1 hour, centrifuge at 13000 r / m for 10 minutes, take the supernatant, add PEG 8000 to a final concentration of 10%, incubate on ice for 3 hours, centrifuge at 13000 r / m for 20 minutes, discard the supernatant, and resuspend the precipitate in 2 mL of SM buffer.

[0046] Third, take 600 μL of the resuspended solution, add 30 μL of proteinase K and 600 μL of CTAB, incubate at 65°C for 10 min, inverting and mixing 3 times during the incubation period, 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) for extraction to remove 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℃ for 30 min, centrifuge at 13000 r / m for 10 min, discard the supernatant, and after the ethanol evaporates, add 120 μL ddH2O to dissolve the phage nucleic acid.

[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 the phylogenetic tree of bacteriophage YL1

[0051] The genome sequence of phage YL1 was compared with existing phage sequences in the NCBI database. A phylogenetic tree was constructed using MEGA 10.0 with *Pseudomonas phage Itty13* as the outgroup. The results showed that phage YL1 and *Ralstonia Phage QkW1* were on the same branch, thus identifying it as belonging to the genus *Brutalophage*. Figure 3 ).

[0052] Example 6

[0053] preservation

[0054] The bacteriophage provided by this invention, named Ralstonia Phage YL1 and classified as Ralstonia Phage, was deposited on October 23, 2024, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M20242304 and address at Wuhan University, Wuhan, China.

[0055] Example 7

[0056] Optimal Multiple of Infection (MOI) for Bacteriophage YL1

[0057] The *Ralstonia solanacearum* culture and phage YL1 prepared in Example 1 were inoculated at an inoculum size of 0.3% (V / V), and the concentration was determined after overnight incubation. Infection complexes with MOIs of 3, 2, 1, 0, -1, -2, and -3 (Table 1) were then incubated at 30°C and the optimal pH for 6 hours. The titer of the phages with different ratios was then determined using the bilayer plate method. Subsequently, lysis curves were measured: equal proportions of phage culture and host were added to 48-well plates, and co-cultured at 30°C for 12 hours. The OD values ​​of the co-culture medium from 0 to 12 hours were measured using a TECAN spark microplate reader. 600 Values ​​were calculated and the rupture curve was plotted.

[0058] Table 1. Phage MOI Ratio Table

[0059]

[0060] The MOI of bacteriophage YL1 was determined, and the results showed that the optimal MOI for bacteriophage YL1 was 2 ( Figure 4 The lysis curve of bacteriophage YL1 against Ralstonia solanacearum was determined according to the optimal MOI ratio. It was found that the OD of the co-culture medium increased within 4 hours after the addition of bacteriophage. 600 The OD value dropped below 0.074, while the control (Ralstonia solanacearum) showed a lower OD value. 600 The value increased continuously over 12 hours, reaching 0.429 at 3 hours and 1.128 at 12 hours. The experimental results indicate that when MOI = 2, the bacteriophage can produce more progeny.

[0061] Example 8

[0062] Determination of the lysis profile of bacteriophage YL1

[0063] Twenty-three strains of *Ralstonia solanacearum* were streaked to activate the bacteria. Single colonies were inoculated into 10 mL of LB broth and incubated overnight at 30°C with shaking at 180 rpm. The overnight-cultured *Ralstonia solanacearum* strains and bacteriophage YL1 culture were then inoculated into 10 mL of LB broth at an equal ratio of 0.5% (V / V). A control group without bacteriophage YL1 culture was used. After co-culturing at 30°C and 180 rpm for 6 hours, the OD values ​​of the treatment and control groups were measured. 600 .

[0064] The lytic activity of bacteriophage YL1 against different Ralstonia solanacearum species is shown in Table 2. The activity was determined by the OD values ​​of the culture medium after lysis. 600 The ratio of the mean to the control (CK) was defined as 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 strain, indicating that phage YL1 has a broad lysis spectrum and high lysis activity.

[0065]

[0066] Table 2. Lysis patterns of bacteriophage YL1 against different Ralstonia solanacearum species (expressed as OD). 600 Value measurement)

[0067]

[0068]

[0069] Example 9: Temperature tolerance of bacteriophage YL1

[0070] Ralstonia solanacearum suspension and bacteriophage YL1 culture medium were prepared according to the above method. Bacteriophage YL1 was incubated in a water bath at 30℃, 37℃, 50℃, 60℃, 70℃, and 80℃ for 1 hour, with each temperature gradient repeated three times. The titer was determined using the double-layer plate method. Figure 5 The results showed that the optimal temperature range was 30–60℃, and the lethal temperature was 80℃.

[0071] Example 10

[0072] The acid and alkali tolerance of bacteriophage YL1

[0073] Prepare *Ralstonia solanacearum* suspension and bacteriophage YL1 culture medium 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, and 13.0 (990 μL) using NaOH or HCl, respectively. Then add 10 μL of bacteriophage YL1 to each solution and incubate at 30°C for 1 h. Repeat the incubation process three times for each pH setting. Determine the titer using the double-layer plate method. Figure 6 The results showed that the activity of phage YL1 remained stable in the pH range of 4–11, indicating that phage YL1 has good tolerance to the above pH range.

[0074] Example 11

[0075] Method of administration of bacteriophage preparations

[0076] The pot experiment included two treatments, with three replicates per treatment and 16 tobacco seedlings per replicate. The OD values ​​of the *Ralstonia solanacearum* solution prepared in Example 1 were first measured.600 Adjust the solution to 0.1 and inoculate each tobacco seedling with 10 mL of Ralstonia solanacearum solution. Separately prepare bacteriophage YL1 culture medium as described above and dilute it to 1×10⁻⁶. 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 cycle was 12h / 12h. Fourteen days after inoculation, the disease severity of each seedling was assessed 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 efficacy of 90.82%, indicating good control efficacy (Table 3). Figure 7 ).

[0077] Table 3 Evaluation of the efficacy of YL1 potted plants

[0078]

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

[0080] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those 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 invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Use of the bacteriophage YL1 in the lysis of Pseudomonas solanacearum, characterized in that, The bacteriophage YL1 is named Ralstonia Phage YL1, and its classification name is Ralstonia Phage, which is preserved in the China Center for Type Culture Collection on October 23, 2024, with a preservation number of CCTCC NO: M 20242304 and a preservation address of Wuhan, Wuhan University, China. The P. solanacearum is P. solanacearum GMI1000.

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