Cracking bacteriophage QKW1 for preventing and controlling tobacco bacterial wilt, biological preparation containing bacteriophage and application

Abstract

The invention relates to the technical field of biological control of bacterial wilt, and discloses a lytic bacteriophage QKW1 for preventing and controlling tobacco bacterial wilt, a biological preparation containing the bacteriophage and application. The collection number of the lytic bacteriophage QKW1 is CCTCC (China Center For Type Culture Collection) NO: M20232698. The preparation method of the biological agent comprises the following steps: inoculating the lysing bacteriophage into a culture solution containing host bacteria, and culturing, wherein the culture is selected from at least one of seed culture, multiplication culture and concentration culture. The method for preventing and treating the plant diseases comprises the following steps: inoculating the biological agent and the biological agent prepared by the method into soil at the stem base of a plant through root irrigation. The lysing bacteriophage can efficiently infect and lyse ralstonia solanacearum, and can be used for biological control of plant bacterial wilt.

Description

Technical Field

[0001] The present invention relates to the technical field of biological control of tobacco bacterial wilt, and in particular to a lytic bacteriophage QKW1 for controlling tobacco bacterial wilt, a biological preparation containing the bacteriophage, and applications thereof. Background Art

[0002] Bacterial wilt is a very common soil-borne disease of crops in the agricultural planting process, caused by infection with Ralstonia solanacearum complex species (referred to as Ralstonia solanacearum complex species). Normally, Ralstonia solanacearum invades plants through the root growth point or physical wound of the plant, then migrates and reproduces from the vascular bundle to the aboveground part, then blocks the vascular bundle, and produces toxic substances to damage the plant, eventually causing the plant to wilt and die. Since Ralstonia solanacearum can infect more than 50 families and more than 200 crops, it is more common and serious to infect Solanaceae crops such as tomatoes, peppers, tobacco, and potatoes. Under natural conditions, Ralstonia solanacearum has a strong variability and produces different specialized types of wilt. In addition, the transmission pathway of Ralstonia solanacearum is complex and the host range is wide, which causes difficulties in prevention and control. In recent years, through chemical agents, disease-resistant varieties planting and crop rotation and other agronomic integrated measures, the harm of bacterial wilt can be reduced to a certain extent, but the occurrence of the disease cannot be effectively controlled.

[0003] At present, biological control has attracted wide attention due to its advantages such as environmental friendliness, safety and low cost, especially bacteriophages, which have attracted wide attention due to their high efficiency, specificity and safety. Studies have found that Ralstonia solanacearum phages can effectively reduce the number of Ralstonia solanacearum in soil in potted plants and field trials. Numerous studies have shown that bacteriophages can not only target and kill the corresponding pathogens, but also have the ability to self-replicate exponentially to prevent and control bacterial infections. Importantly, bacteriophages of corresponding bacterial hosts are ubiquitous in nature, easy to separate and have a short purification cycle, stable titer, easy to store, transport and use.

[0004] Although the research on the use of bacteriophages to control bacterial diseases is very extensive, its lytic phages are widely concerned and applied due to their host specificity, high lysis efficiency, no toxic side effects, and high safety, especially in the aquaculture and animal husbandry industries in recent years. However, due to the good efficacy and application effect of phages, high-titer phage preparations are often required to ensure it. However, the existing phage production methods are difficult, inefficient, and have poor phage activity, which hinders the development of related industries in phage production and application.

[0005] Therefore, providing a bacteriophage that can efficiently infect and lyse bacterial wilt pathogens and a method for preparing a high-titer active preparation thereof is of great significance for preventing and controlling soil-borne bacterial wilt diseases of crops. Summary of the invention

[0006] The purpose of the present invention is to overcome the problems existing in the prior art and to provide a lytic bacteriophage QKW1 for preventing and controlling tobacco bacterial wilt, a biological preparation containing the phage and its application. The lytic phage can efficiently infect and lyse Ralstonia solanacearum and can be used for biological control of plant bacterial wilt.

[0007] In order to achieve the above object, the present invention provides a lytic bacteriophage in a first aspect. The deposit number of the lytic bacteriophage (Ralstonia solanacearum phage QKW1) is CCTCC M20232698.

[0008] The second aspect of the present invention provides a biological preparation, which contains the above-mentioned lytic bacteriophage.

[0009] Preferably, the biological preparation is a solid preparation or a liquid preparation.

[0010] Preferably, the concentration of lytic phage in the biological preparation is 2×10 6 -3×10 13 PFU / mL, more preferably 10 12 -10 13 PFU / mL.

[0011] The third aspect of the present invention provides a method for preparing a biological agent, which comprises: inoculating a lytic phage into a culture solution containing a host bacterium for culturing, wherein the culturing is selected from at least one of seed culture, proliferation culture and concentrated culture.

[0012] Preferably, the host bacteria is Ralstonia solanacearum tian (deposited with the same accession number as QKW1).

[0013] Preferably, the culture solution contains glucose, beef extract, peptone and yeast powder.

[0014] Preferably, the culture solution contains 8-12 g / L glucose, 3-7 g / L peptone, 2-4 g / L beef extract, and 0.5-1.5 g / L yeast powder.

[0015] Preferably, the seed culture conditions include: temperature of 25-30°C, rotation speed of 800-1000rpm, and time of 12-20h;

[0016] The conditions of the proliferation culture include: temperature of 25-30°C, rotation speed of 150-200 rpm, and time of 20-30 hours;

[0017] The concentrated culture process includes: subjecting the proliferation liquid obtained by the proliferation culture to solid-liquid separation I to obtain a proliferated phage liquid, and mixing the proliferated phage liquid with the cells of the host bacteria and then culturing I.

[0018] Preferably, the conditions of the culture I include: temperature of 25-30°C, rotation speed of 150-200 rpm, and time of 4-8 hours.

[0019] Preferably, the preparation method further comprises: performing solid-liquid separation II on the culture obtained from at least one of the seed culture, proliferation culture and concentration culture to obtain the culture supernatant as the biological agent.

[0020] The fourth aspect of the present invention provides the use of the above-mentioned lytic bacteriophage, the above-mentioned biological agent, and the biological agent prepared by the above-mentioned method in preventing and controlling plant diseases.

[0021] Preferably, the plant disease is bacterial wilt, more preferably tobacco bacterial wilt.

[0022] A fifth aspect of the present invention is a method for preventing and controlling plant diseases, which comprises the following steps: inoculating the above-mentioned biological agent or the biological agent prepared by the above-mentioned method into the soil at the base of the plant stem by root irrigation.

[0023] Preferably, the amount of the lytic phage added is 10 7 -10 9 PFU / strain.

[0024] Through the above technical scheme, the beneficial effects of the present invention are as follows: the lytic bacteriophage provided by the present invention can not only efficiently infect the lytic host bacterial wilt bacteria, but also has high activity in the temperature range of 4-50°C and pH range of 5-10, strong lytic ability, and can show strong bactericidal ability. In a potted plant experiment using the lytic bacteriophage provided by the present invention to prevent and control soil-borne bacterial wilt, it was found that the prevention effect of tobacco bacterial wilt can reach 80.96%, which can effectively prevent and control the occurrence of tobacco bacterial wilt from the root, and can be used to prepare biological preparations for preventing and controlling tobacco bacterial wilt.

[0025] Other features and advantages of the present invention will be described in detail in the following detailed description.

[0026] Biological Deposit

[0027] The strain provided by the present invention is a lytic phage QKW1 (Ralstonia solanacearum phageQKW1), which was deposited in the China Center for Type Culture Collection (address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, postal code: 430072, the abbreviation of the depository unit is CCTCC) on December 27, 2023, and the deposit number is CCTCC NO: M20232698. The host of the lytic phage QKW1, the bacterial wilt pathogen Ralstonia solanacearumtian, is preserved with the phage under the same deposit number. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The morphological diagram of the bacterial colony of Ralstonia solanacearum tian in Example 1;

[0029] Figure 2 The plaque morphology of bacteriophage QKW1 on a plate in Example 2;

[0030] Figure 3 This is an electron microscopic morphology of bacteriophage QKW1 in Example 3;

[0031] Figure 4 The figure is the genome information diagram of the bacteriophage QKW1 in Example 4, wherein, from the inside to the outside, the innermost black circle represents the size and logo of the genome; the purple-green circle with a peak represents GC-skew (GC skew = (GC) / (G+C)), green represents GC-skew greater than 0, and purple represents GC-skew less than 0, which can be used to determine the replication origin of circular DNA; the black circle with a peak represents the GC content: the peak outward (inward) indicates that the GC content of the region is higher (lower) than the average GC content of the whole genome, and the higher the peak, the greater the difference with the average GC content; the outer dark blue circle represents the CDS sequence of the QKW1 genome, and the direction, length and position of the arrows respectively represent the direction, size and position of the CDS sequence on the genome;

[0032] Figure 5 This is a graph showing the thermal stability potency of bacteriophage QKW1 in Example 5;

[0033] Figure 6 This is the pH tolerance stability titer diagram of bacteriophage QKW1 in Example 5;

[0034] Figure 7 This is a diagram showing the potted plant test results of bacteriophage QKW1 inhibiting the growth of Ralstonia solanacearum in Example 6. DETAILED DESCRIPTION

[0035] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0036] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0037] The first aspect of the present invention provides a lytic bacteriophage, wherein the deposit number of the lytic bacteriophage (Ralstoniasolanacearum phage QKW1) is CCTCC NO: M20232698.

[0038] The lytic phage provided by the present invention is isolated from fresh rhizosphere soil of tobacco with bacterial wilt collected from a tobacco field (the collection location is Changsha City, Hunan Province). The isolation of the lytic phage can adopt a conventional method for phage isolation in the art, for example, a mixed enrichment method of "bacterial liquid + soil + culture medium" can be adopted.

[0039] The inventors of the present invention screened out a bacteriophage QKW1 that can efficiently infect the lytic host bacterial wilt pathogen, and performed DNA extraction and identification. The identification results showed that the head of the phage was a regular hexahedral structure with a long tail; the whole genome sequence was similar to Ralstonia phage p2110 (OP947226, coverage 78%, consistency 96.93%) and Ralstonia phageRPZH6 (MT361768, coverage 81%, consistency 94.96%). Combined with the results of electron microscopic morphological identification, it was determined that phage QKW1 belongs to the Caudovirales order, Siphoviridae family, and was named lytic phage QKW1 (Ralstonia solanacearum phage QKW1), which was deposited in the China Center for Type Culture Collection on December 27, 2023, with the deposit number CCTCC NO:M20232698, the storage address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0040] The lytic phage provided by the present invention can produce a large number of phages with biological activity after culture. The present invention has no particular limitation on the culture method, as long as the lytic phage can be proliferated in large quantities by the culture method. For example, a live sample of the lytic phage can be inoculated into the culture system of the host bacteria, and after culture at a temperature of 25-30°C, solid-liquid separation is performed to obtain a culture solution containing phage. Among them, the culture medium can be a culture medium conventionally used in the art, for example, the culture medium formula can be 8-12 g / L of glucose, 3-7 g / L of peptone, 2-4 g / L of beef extract, and 0.5-1.5 g / L of yeast powder (referred to as NA liquid culture medium).

[0041] In the present invention, solid-liquid separation can be achieved by centrifugation and / or filtration. The conditions of the centrifugation and filtration can be conventional conditions in the art, which are well known to those skilled in the art and will not be described in detail here.

[0042] A second aspect of the present invention provides a biological preparation, which contains the lytic bacteriophage according to claim 1.

[0043] According to the present invention, there is no particular limitation on the dosage form of the biological preparation. It can be prepared into different dosage forms according to different intended uses, and corresponding excipients and other ingredients can be added. For example, the biological preparation can be a liquid preparation (for example, a culture solution containing lytic bacteriophages) and / or a solid preparation (for example, a powder prepared by drying lytic bacteriophages or a high-purity preparation prepared by separation and purification steps). Among them, what kind of excipients to add to what kind of dosage form is well known to those skilled in the art and will not be described in detail here.

[0044] In the present invention, preferably, the biological preparation is a liquid preparation, and the concentration of the lytic bacteriophage in the biological preparation can be specifically selected according to the specific situation. Preferably, the concentration of the lytic bacteriophage in the biological preparation is 2×10 6 -3×10 13 PFU / mL, more preferably 10 12 -10 13 PFU / mL.

[0045] The lytic bacteriophage and corresponding biological agent provided by the present invention can not only efficiently infect the lytic host bacterial wilt bacteria, but also have high activity in the temperature range of 4-50°C and pH range of 5-10, strong lytic ability, and can show strong bactericidal ability. In a potted plant experiment using the lytic bacteriophage provided by the present invention to prevent and control soil-borne bacterial wilt, it was found that the prevention effect of tobacco bacterial wilt can reach 80.96%, which can effectively prevent and control the occurrence of tobacco bacterial wilt from the root.

[0046] The third aspect of the present invention provides a method for preparing a biological agent, which comprises: inoculating a lytic phage into a culture solution containing a host bacterium for culturing, wherein the culturing is selected from at least one of seed culture, proliferation culture and concentrated culture.

[0047] In the present invention, seed culture, proliferation culture and concentration culture are cultivation processes that can be performed sequentially. The cultivation can be seed culture, and the seed liquid obtained by seed culture is subjected to solid-liquid separation to obtain a culture supernatant containing lytic phage as the biological preparation; the cultivation can also be proliferation culture after seed culture, and the proliferation liquid obtained by proliferation culture is subjected to solid-liquid separation to obtain a culture supernatant containing lytic phage as the biological preparation; the cultivation can also be seed culture, proliferation culture and concentration culture in sequence, and the concentrated liquid obtained by concentration culture is subjected to solid-liquid separation to obtain a culture supernatant containing lytic phage as the biological preparation.

[0048] According to the present invention, preferably, the host bacteria is Ralstonia solanacearum, wherein Ralstonia solanacearum can be purchased commercially or isolated by oneself.

[0049] In the present invention, Ralstonia solanacearum tian is used, and the inventor collects tobacco bacterial wilt disease plant samples from tobacco fields in the tobacco leaf producing area of ​​Changsha, Hunan Province, soaks fresh stems infected with bacterial wilt disease with sterile water to take out bacterial pus suspension, and uses NA solid culture medium plates containing TTC (red tetrazolium, the dosage is 50 mg / kg; filtered and sterilized) to streak and select single colonies, and after purifying the single colonies 3-5 times, the obtained colonies are obtained by 27F / 1492R sequencing and identification.

[0050] In the present invention, the host bacteria in the culture solution containing the host bacteria are Ralstonia solanacearum in the logarithmic growth phase; the Ralstonia solanacearum in the logarithmic growth phase can be obtained by a conventional culture method in the art.

[0051] According to the present invention, the culture solution can adopt a conventional liquid culture medium, so as to provide the nutrients required for the growth and proliferation of Ralstonia solanacearum and bacteriophage. Preferably, the culture solution contains glucose, beef extract, peptone and yeast powder. Exemplarily, the culture solution contains 8-12g / L of glucose, 3-7g / L of peptone, 2-4g / L of beef extract, and 0.5-1.5g / L of yeast powder (referred to as NA liquid culture medium). The inventors have found that under this preferred specific embodiment, the growth rate of the host bacteria and the bacteriophage can be increased, and the production efficiency of the biological preparation can be improved.

[0052] In the present invention, the lytic phage can be activated and cultured before seed culture. For example, the frozen lytic phage mother solution is taken out and inoculated into a fresh logarithmic phase culture solution containing host bacteria, and cultured for 12-20 hours at a temperature of 25-30°C and a rotation speed of 150-200rpm, and then centrifuged to obtain a supernatant, and the supernatant is inoculated with host bacteria on a double-layer plate, and inverted overnight culture is carried out at a temperature of 25-30°C. After selecting a clear single plaque on the double-layer plate cultured overnight and inoculating it, the seed culture is carried out.

[0053] In the present invention, the proliferation culture process includes: after solid-liquid separation of the culture of the seed culture, inoculating the culture supernatant into a new culture system (for example, NA liquid culture medium containing about 1 volume % host bacteria), and then performing the proliferation culture.

[0054] According to the present invention, preferably, the conditions for the seed culture include: a temperature of 25-30°C (using a constant temperature metal bath), a rotation speed of 800-1000 rpm, and a time of 12-20 hours; the conditions for the proliferation culture include: a temperature of 25-30°C (using a ventilation shaker), a rotation speed of 150-200 rpm, and a time of 20-30 hours. The inventors have found that under this preferred specific embodiment, the growth rate of the host bacteria and the phage can be increased, thereby improving the production efficiency of the biological preparation.

[0055] According to the present invention, preferably, the concentrated culture process comprises: subjecting the proliferation liquid obtained by the proliferation culture to solid-liquid separation I to obtain a proliferated phage liquid, and mixing and suspending the proliferated phage liquid and the host bacteria to sufficient mixing and culturing I. The host bacteria can be inoculated into NA liquid culture medium for fermentation culture, and OD 600 When the pH value is about 1.0-4.0, the fermentation broth is centrifuged at a low speed and low temperature to collect live host bacteria. According to the present invention, preferably, the conditions of the culture I include: a temperature of 25-30°C (using a ventilation shaker), a rotation speed of 150-200rpm, and a time of 4-8h. The inventors have found that under this preferred specific embodiment, the growth rate of the host bacteria and the phage can be increased. According to the present invention, preferably, the preparation method also includes: performing solid-liquid separation II on the culture obtained from at least one of the seed culture, proliferation culture and concentration culture to obtain the culture supernatant as the biological agent.

[0056] In the present invention, the solid-liquid separation II can be carried out by combining centrifugation with membrane filtration to obtain a high-titer biological preparation containing the lytic bacteriophage.

[0057] Exemplarily, the concentrated culture process includes: centrifuging the proliferation liquid obtained from the proliferation culture at 10000-13000rpm for 5-10min to obtain a phage supernatant; using 0.5-3 times the volume of the phage supernatant liquid of the host bacteria Ralstonia solanacearum tian for 30-36h liquid fermentation culture, centrifuging at 3000-4500rpm for 5-10min to obtain a bacterial precipitate; then suspending the host bacteria bacterial precipitate with the phage supernatant sufficiently, culturing at 25-30°C, on an aerated shaker, at a speed of 150-200rpm for 4-8h, and after the cultivation is completed, centrifuging at 10000-13000rpm for 5-10min and passing through a 0.22um filter membrane, the resulting filtrate is the phage concentrated liquid, and a high-titer biological preparation containing the lytic phage is obtained.

[0058] The fourth aspect of the present invention provides the use of the above-mentioned lytic bacteriophage, the above-mentioned biological agent, and the biological agent prepared by the above-mentioned method in preventing and controlling plant diseases.

[0059] According to the present invention, preferably, the plant disease is bacterial wilt, more preferably tobacco bacterial wilt.

[0060] A fifth aspect of the present invention is a method for preventing and controlling plant diseases, which comprises the following steps: inoculating the above-mentioned biological agent or the biological agent prepared by the above-mentioned method into the soil at the base of the plant stem by root irrigation.

[0061] According to the present invention, preferably, the amount of the lytic phage added is 10 7 -10 9 PFU / strain; the inventors found that under this preferred specific implementation mode, the ability to prevent tobacco bacterial wilt can be improved.

[0062] The present invention will be described in detail below through examples.

[0063] In the following examples, unless otherwise specified, the raw materials and reagents used are conventional commercially available products.

[0064] In the following examples, the preparation method of NA liquid culture medium is as follows: 10 g / L glucose, 5 g / L peptone, 3 g / L beef extract, 1 g / L yeast powder, 1 L deionized water, natural pH, 121°C, sterilized for 20 min and cooled before use;

[0065] The preparation method of NA solid culture medium is: glucose 10g / L, peptone 5g / L, beef extract 3g / L, yeast powder 1g / L, agar 15g / L, deionized water 1L, natural pH, 121℃, sterilize and cool for 20 minutes before use.

[0066] Example 1 Screening of soil-borne pathogens of Ralstonia solanacearum

[0067] Tobacco bacterial wilt samples were collected from tobacco fields in the tobacco production area of ​​Changsha, Hunan Province. Fresh stems infected with bacterial wilt were soaked in sterile water to obtain bacterial pus suspension. Single colonies were selected by streaking on NA solid medium plates containing TTC (red tetrazolium, dosage was 50 mg / kg; filtered and sterilized). After single colonies were purified 3-5 times, 27F / 1492R sequencing was used for identification to obtain pure strains of Ralstonia solanacearum tian, see Figure 1 The partial sequence fragment of 16S rDNA is shown in SEQ ID NO.1.

[0068] The strain Ralstonia solanacearum tian was inoculated into NA liquid medium for fermentation culture to obtain the host bacteria Ralstonia solanacearum tian bacterial liquid in the logarithmic phase.

[0069] Example 2 Screening and purification of soil-borne pathogenic bacteriophage of Ralstonia solanacearum

[0070] The strain Ralstonia solanacearum tian isolated in Example 1 was used as a host, and fresh soil from the rhizosphere of tobacco in a tobacco field with bacterial wilt was collected from a tobacco field to isolate the bacteriophage; specifically, a mixed enrichment method of "bacterial solution + soil + culture medium" was adopted, in a 250ml sterile conical flask, 100ml of sterile liquid NA culture medium, 2ml of fresh logarithmic phase host bacteria Ralstonia solanacearum tian bacterial solution, 2g of fresh soil from the above sources were added, and the mixture was placed at 28°C and cultured on a shaking table at 180rpm for 16h, then allowed to stand and the supernatant was taken, centrifuged at a speed of 12000rpm for 10min and filtered through a 0.22μm filter membrane, the resulting supernatant was the phage stock solution, and the stock solution was then washed with sterile water at 10 -1 -10 -7 Dilute and spread on double-layer NA solid plates containing host bacteria Ralstoniasolanacearumtian to screen phage colonies. Figure 2 .

[0071] Purification of phage colonies: A single phage plaque on a double-layer NA solid plate was picked up and inoculated into a new NA liquid culture system (100 ml NA culture medium + 1 ml fresh host bacterial liquid), and cultured overnight at 28°C and 180 rpm in a shaking incubator. The culture was then centrifuged at 12,000 rpm for 10 min and filtered through a 0.22 μm filter membrane to obtain a filtrate. After picking a single plaque and inoculating and culturing the plate 3-5 times, the filtrate and / or phage in the plate was recorded as phage QKW1, and the filtrate was the mother liquor.

[0072] Example 3 Electron microscopic observation of bacteriophage QKW1

[0073] The phage QKW1 mother solution in Example 2 was added to 10 ml of the host bacteria Ralstonia solanacearum tian in the logarithmic growth period at 1% to 5% by volume, and cultured in a shaking incubator at 28°C and 180 rpm for 16 h. The mixture was centrifuged at 12000 rpm for 10 min and filtered through a 0.22 μm filter membrane to obtain a phage filtrate. The filtrate titer was determined by the dilution method. The filtrate was diluted with sterile water to a phage concentration of 10 8 PFU / mL, and then conduct electron microscopy (TEM) observation. Take 20μl of phage filtrate on the copper mesh, let it stand for 15 minutes, use filter paper to absorb excess liquid on the side, add a drop of 2% tungsten phosphate solution on the copper mesh to dye it, let it stand for 10 minutes, use filter paper to absorb excess staining liquid on the side again, let it stand for 10 minutes to dry the sample, and observe the morphology of phage under electron microscope. The results are as follows Figure 3 As shown, the head of bacteriophage QKW1 has a regular hexahedral structure with a long tail, and it was preliminarily identified as a long-tailed phage.

[0074] Example 4 Whole genome analysis of bacteriophage QKW1

[0075] The genomic DNA of bacteriophage QKW1 was extracted using a phage DNA extraction kit according to the operating steps and sent to Shanghai Paisono Company for sequencing. The sequencing results showed that the genome of bacteriophage QKW1 was a circular double-stranded DNA with a total of 73 ORFs and a total length of 59889 bp (part of the representative ORF gene sequences in the whole genome sequence are shown in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4), the GC content was 64.55%, and the contents of A, G, C, and T bases were 19.42%, 33.14%, 31.41%, and 16.02%, respectively. The genome information diagram is shown in Figure 4 .

[0076] The results of genome comparison showed that phage QKW1 was similar to Ralstonia phage p2110 (OP947226, coverage 78%, consistency 96.93%) and Ralstonia phage RPZH6 (MT361768, coverage 81%, consistency 94.96%). Combined with the results of electron microscopy morphological identification, it was determined that phage QKW1 belonged to the Caudovirales, Siphoviridae, and was named lytic phage QKW1 (Ralstonia solanacearumphage QKW1). It was deposited in the China Center for Type Culture Collection on December 27, 2023, with the deposit number CCTCC NO: M20232698, and the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0077] Example 5 Preparation of high titer biological preparation of bacteriophage QKW1 and determination of biological characteristics

[0078] (1) 1 ml of the frozen bacteriophage QKW1 stock solution obtained in Example 2 was taken out of the refrigerator and inoculated into 100 ml of fresh NA liquid culture medium of the host bacteria Ralstonia solanacearum tian in the logarithmic phase at 1% by volume. After culturing at 28° C. and 180 rpm for 16 h, the supernatant was centrifuged at 13,000 rpm for 5 min, and the supernatant was spotted on a double-layer NA plate of the host bacteria and incubated inverted overnight at 28° C.;

[0079] (2) Select a clear single plaque on the above-mentioned double-layer plate cultured overnight, pick up a single plaque with a sterile toothpick and inoculate it into 1 ml of NA liquid culture medium, and inoculate 1% by volume of fresh logarithmic phase host bacteria Ralstonia solanacearum tian culture medium, and culture it at 28°C, 900 rpm constant temperature metal bath conditions for 16 hours; centrifuge the culture medium at a speed of 12000 rpm for 5 minutes and then filter it through a 0.22 μm filter membrane. The filtrate is the phage QKW1 seed liquid;

[0080] (3) Then, the phage seed solution and the host bacteria Ralstonia solanacearum tian solution were inoculated into NA liquid culture medium at 1% by volume, cultured in a constant temperature shaker at 28°C and 180 rpm for 24 h, centrifuged at 12000 rpm for 5 min, filtered through a 0.22 μm filter membrane, and the filtrate was the phage QKW1 proliferation medium;

[0081] (4) Using NA liquid medium to ferment the host bacteria Ralstonia solanacearum tian, wait until OD 600When the pH value is about 1.0-4.0, the host bacteria fermentation liquid is collected and centrifuged at a speed of 3500 rpm and a temperature of 4°C for 5-10 minutes to obtain the host bacteria cells;

[0082] (5) using 0.5 times the volume of the fermentation liquid from step (4) and the fresh phage proliferation liquid obtained from step (3) to suspend the host bacterial cell precipitate obtained from step (4) for 3-10 minutes, mixing thoroughly, and incubating in a constant temperature shaker at 28° C. and 180 rpm for 6 hours;

[0083] (6) The culture solution obtained in step (5) was centrifuged at 12000 rpm and room temperature for 5 min, and filtered through a 0.22 μm filter membrane. The filtrate obtained was the phage QKW1 concentrate.

[0084] The phage seed solution, proliferation solution and concentrated solution in Example 5 were tested for titer respectively and immediately by double-layer plate method. The titer testing process is as follows: the phage filtrate liquid sample to be tested was taken from 10 -1 to 10 -13 Perform 10-fold gradient dilution and titer determination. Take 100 μl of phage dilution solution and 100 μl of host bacteria Ralstonia solanacearum tian bacterial solution to make a double-layer plate. 8 The double-layer plate with CFU / mL host bacteria Ralstonia solanacearumtian was used as the control, and the culture was kept at a constant temperature of 28℃ overnight. The phage titer of the phage filtrate was calculated by observing the number of plaques and multiplying it by the dilution multiple. The results are shown in Table 1. The seed liquid, proliferation liquid and concentrated liquid in the preparation process of the biological preparation containing Ralstonia solanacearum phage QKW1 all showed a certain titer, among which the phage QKW1 concentrated liquid was a high-titer phage biological preparation.

[0085] The titer results of the phage seed solution, proliferation solution and concentrated solution showed a large gradient, reflecting the importance of each step. The final concentrated solution can be as high as 10 13 PFU / ml, which reflects a better concentration preparation effect, which is compared with the current conventional phage biological preparations (generally 10 8-10 PFU / ml), the titer was greatly improved.

[0086] Table 1 Potency determination of bacteriophage QKW1 biological preparation

[0087] sample Titer (PFU / ml) Phage QKW1 seed solution <![CDATA[2.9×10 6 ±4.7×10 5 ]]> Bacteriophage QKW1 proliferation medium <![CDATA[3.1×10 10 ±7.5×10 9 ]]> Bacteriophage QKW1 concentrate <![CDATA[1.9×10 13 ±0.9×10 13 ]]>

[0088] Test to determine the temperature and pH tolerance of bacteriophage QKW1 biological preparation.

[0089] The phage QKW1 concentrate was adjusted to a titer of 10 with sterile water.10 PFU / ml was used as the test solution to determine the temperature and pH tolerance. The temperature range was -20, 4, 28, 37, 50, 60, and 70°C, and the pH range was 3-12. The temperature test process was as follows: the bacteriophage QKW1 test solution was placed on a constant temperature shaker for incubation for 1 hour, and the titer was determined after cooling to room temperature. The pH test process was as follows: the solution was adjusted to different pH values ​​with 0.1M HCL or NaOH, and then incubated for 1 hour, and then adjusted to pH = 7 with SM buffer, and then the titer was determined. The results are shown in Table 1. Figure 5 and Figure 6 shown.

[0090] Figure 5 and Figure 6 The results of the analysis showed that the prepared bacteriophage QKW1 concentrate had very strong thermal stability and acid-base tolerance, with the best effect when the thermal tolerance range was below 50°C, the best effect when the pH tolerance range was 5-10, and the titer hardly decreased. This shows that the high-titer bacteriophage QKW1 biological preparation prepared by the production method provided by the present invention has strong activity and high stability.

[0091] Example 6 Greenhouse tobacco potted bacterial wilt prevention and control test

[0092] A greenhouse tobacco seedling pot experiment was used to detect the ability of bacteriophage QKW1 to prevent and control tobacco bacterial wilt disease.

[0093] The test soil was used as the seedling medium, and the tobacco variety was Yunyan 87. Four treatment groups were set up in the experiment, as shown in Table 2.

[0094] Table 2 Grouping Processing

[0095]

[0096] Each treatment group had 3 parallels, each with 15 seedlings. The tobacco seedlings were transplanted when they had 4 leaves. Seven days after transplanting, they were inoculated with Ralstonia solanacearum tian, and two days later, they were inoculated with bacteriophage QKW1. Then they were cultured at 30℃, 6000lm of light, 60-80% of humidity, 14h of light / 10h of darkness. On the 30th day after inoculation, the incidence and disease index of tobacco seedling bacterial wilt were investigated, and the control effect was calculated. The incidence, disease index and relative control efficiency of tobacco bacterial wilt were investigated and calculated according to the standard "GB / T23222-2008 Grading and Investigation Methods of Tobacco Pests and Diseases". The final results are shown in Table 3, and the pot test effect diagram is shown in Figure 7 .

[0097] The results in Table 3 show that the T3 group treated with bacteriophage QKW1 after inoculation with bacterial wilt pathogenic bacteria had a very significant prevention effect on the incidence of tobacco seedling bacterial wilt disease, with the incidence rate reduced from 84.44% to 17.78%, p<0.05; the disease index reduced from 61.2 to 11.6, p<0.05; the prevention efficiency reached 80.96%, and the effective period lasted for 30 days.

[0098] Table 3 Incidence rate, disease index and control effect of tobacco bacterial wilt in each treatment group

[0099] Treatment Group deal with Effectiveness% Disease Index Incidence % T1 CK - 0c 0c T2 Rstian - 61.2±4.2a 84.44±3.85a T3 Rstian+QKW1 80.96±5.46 11.6±3.3b 17.78±3.85b

[0100] The values ​​in Table 3 are mean ± standard deviation, and the lowercase letters after the values ​​indicate LSD analysis p < 0.05.

[0101] The above results show that the bacteriophage QKW1 isolated from tobacco wilt soil samples collected from tobacco fields in the tobacco producing area of ​​Changsha, Hunan Province, has a high bactericidal activity against tobacco wilt Ralstonia solanacearum tian, and can effectively solve the problem of bacterial wilt prevention and control in crop cultivation in the region.

[0102] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A lytic bacteriophage, characterized in that The deposit number of the lytic phage (Ralstonia solanacearumphage QKW1) is CCTCC NO: M20232698.

2. A biological agent, characterized in that: The biological preparation contains the lytic bacteriophage according to claim 1.

3. The preparation according to claim 2, characterized in that The biological preparation is a solid preparation or a liquid preparation; Preferably, the concentration of lytic phage in the biological preparation is 2×10 6 -3×10 13 PFU / mL, more preferably 10 12 -10 13 PFU / mL.

4. A method for preparing a biological agent, characterized in that: The preparation method comprises: inoculating a lytic phage into a culture solution containing host bacteria for culturing, wherein the culturing is selected from at least one of seed culture, proliferation culture and concentrated culture.

5. The preparation method according to claim 4, characterized in that: The host bacteria is Ralstonia solanacearum tian.

6. The preparation method according to claim 4 or 5, characterized in that: The culture solution contains glucose, beef extract, peptone and yeast powder; Preferably, the culture solution contains 8-12 g / L glucose, 3-7 g / L peptone, 2-4 g / L beef extract, and 0.5-1.5 g / L yeast powder.

7. The preparation method according to claim 4 or 5, characterized in that: The seed culture conditions include: temperature of 25-30°C, rotation speed of 800-1000rpm, and time of 12-20h; The conditions of the proliferation culture include: temperature of 25-30°C, rotation speed of 150-200 rpm, and time of 20-30 hours; The concentrated culture process comprises: subjecting the proliferation liquid obtained by the proliferation culture to solid-liquid separation I to obtain a proliferated phage liquid, and mixing the proliferated phage liquid with the host bacteria to culture I; Preferably, the conditions of the culture I include: temperature of 25-30°C, rotation speed of 150-200 rpm, and time of 4-8 hours.

8. The preparation method according to claim 4 or 5, characterized in that: The preparation method further comprises: performing solid-liquid separation II on the culture obtained from at least one of the seed culture, proliferation culture and concentration culture to obtain a culture clear liquid.

9. Use of the lytic bacteriophage according to claim 1, the biological agent according to claim 2 or 3, or the biological agent obtained by the method of any one of claims 4 to 8 in preventing and controlling plant diseases; Preferably, the plant disease is bacterial wilt, more preferably tobacco bacterial wilt.

10. A method for preventing and controlling plant diseases, characterized in that: The method comprises the following steps: inoculating the biological agent according to claim 2 or 3 or the biological agent obtained by any one of claims 4 to 8 into the soil at the base of the plant stem by root irrigation; Preferably, the amount of the lytic phage added is 10 7 -10 9 PFU / strain.

Images