CN112831475A

CN112831475A - Lytic bacteriophage and application thereof in prevention and control of soil-borne bacterial wilt of tobacco

Info

Publication number: CN112831475A
Application number: CN202110076959.3A
Authority: CN
Inventors: 韦中; 王硕; 杨天杰; 王孝芳; 王佳宁; 徐阳春; 沈其荣
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2021-01-20
Filing date: 2021-01-20
Publication date: 2021-05-25
Anticipated expiration: 2041-01-20
Also published as: CN112831475B

Abstract

The invention discloses a lytic bacteriophage FQ44, which is classified and named as Laurella (Ralstoniasolana cerarum phage) of solanaceae, and is preserved in China center for type culture Collection in 29 months 9 and 2020, wherein the preservation number of the bacteriophage is CCTCC NO: m2020560. The invention also discloses application of the bacteriophage FQ44 in preventing and controlling tobacco soil-borne bacterial wilt, and the bacteriophage is applied to soil by adopting a root irrigation method. The invention inhibits the tobacco soil-borne bacterial wilt by using the bacterial wilt obligate bacteriophage, thereby avoiding the damage to the common microbial community in the soil while preventing and controlling the soil-borne bacterial wilt. Indoor related experiments show that the bacteriophage FQ44 belongs to Myocapnoraceae, can effectively inhibit the growth of part of ralstonia solanacearum, and has the best effect when applied according to the optimal multiplicity of infection (MOI 1-10); the field experiment effect also shows that the compound enzyme can effectively inhibit the occurrence of bacterial wilt and has wide prospect.

Description

Lytic bacteriophage and application thereof in prevention and control of soil-borne bacterial wilt of tobacco

Technical Field

The invention belongs to the technical field of microorganisms, relates to a bacteriophage and application thereof in prevention and control of tobacco bacterial wilt, and particularly relates to a lytic bacteriophage FQ44 and application thereof in prevention and control of tobacco soil-borne bacterial wilt.

Background

The tobacco bacterial wilt is a soil-borne disease caused by Laurella solanacearum (Ralstonia solanacearum for short), is frequently generated in tobacco planting areas in China, can cause irreversible wilting and death of tobacco, causes serious yield reduction of the tobacco, and restricts the development of tobacco economy. By applying pesticides or fumigating and other traditional modes, the soil-borne ralstonia solanacearum can be killed in a short time, and the occurrence of diseases is effectively slowed down. However, the above method is likely to induce drug resistance of pathogenic bacteria, kill indigenous microorganisms in the soil, destroy soil microbial homeostasis, pollute the environment and affect tobacco quality.

Bacteriophage is a virus that specifically infects bacteria and is ubiquitous in nature. The lytic phage can lyse host cells after infecting the bacteria and affect the bacteriaMetabolism, and effective control of host bacteria. The therapeutic approach to treat pathogenic bacterial infections using bacteriophage lytic bacteria is called phage therapy^[1]. The phage therapy has the advantages of specificity, high efficiency and the like which are incomparable with other antibacterial agents, and gradually becomes one of the research hotspots for developing novel medicines.

Therefore, the method has wide application prospect in preventing and controlling the tobacco bacterial wilt by cracking pathogenic bacteria through the bacteriophage.

Disclosure of Invention

The invention aims to separate and obtain the bacteriophage capable of specifically cracking the tobacco ralstonia solanacearum, detect the basic biological characteristics of the bacteriophage, including plaque size, host range, electron microscope shape, optimal infection complex number and one-step growth curve, detect the indoor bacteriostasis effect of the bacteriophage on the tobacco ralstonia solanacearum and the field control effect of the bacteriophage on the tobacco ralstonia solanacearum, and provide a theoretical basis for developing a novel and efficient preparation for resisting the tobacco ralstonia solanacearum bacteriophage. The inventor finds that: in an indoor experiment, the ralstonia solanacearum obligate phage can specifically crack ralstonia solanacearum separated from tobacco rhizosphere soil and can also crack part of pathogenic ralstonia solanacearum from other soil-crop systems; in field experiments, the bacterial wilt of tobacco can be effectively prevented after the application of the phage.

The purpose of the invention is realized by the following technical scheme:

a lytic bacteriophage FQ44, classified and named as Laurella sp (Ralstonia solanacearum phage), is preserved in China center for type culture Collection in 29 months 9 and 2020, and has a preservation number of CCTCC NO: m2020560.

Lytic phage FQ44 belongs to the order urophages, family Myoviridae (Myoviridae).

The host bacterium of the bacteriophage FQ44 is YNYC-23, is classified and named as Laurella (Ralstonia solanacearum), is preserved in China center for type culture Collection in 29 months 9 and 2020, and has the strain preservation number of CCTCC NO: m2020559.

The invention also aims to provide application of the lytic bacteriophage FQ44 in prevention and control of tobacco soil-borne bacterial wilt.

Preferably, the bacteriophage is applied to the soil at the root of the tobacco by a root irrigation method.

Preferably, the final titer of the phage is 1X 10⁷～1×10⁸PFU/g soil.

The invention also aims to provide a phage preparation for preventing and controlling the soil-borne bacterial wilt of tobacco, wherein the phage preparation comprises the lytic phage FQ44 with the titer of 1 x 10⁸～1×10⁹PFU/mL。

The phage inoculum is prepared by the following method: culturing with NA liquid culture medium to obtain Ralstonia solanacearum liquid in logarithmic growth phase, and culturing Ralstonia solanacearum liquid OD with sterile water₆₀₀Adjusting the value to 0.45-0.55; regulation of phage titer to 1X 10 with sterile water⁸～1×10⁹PFU/mL, placed at OD according to the optimal multiplicity of infection (MOI 1-10)₆₀₀Performing shaking culture at 30 ℃ and 150-180 rpm for 9-12 h in a bacterial solution of ralstonia solanacearum with the value of 0.45-0.55, centrifuging at 12000-14000 rpm for 3min, taking supernatant, and filtering with a 0.22 mu m filter membrane; regulation of phage titer to 1X 10 with sterile water⁸～1×10⁹PFU/mL to obtain the phage inoculum.

The ralstonia solanacearum bacterial liquid is prepared by the following method: activating the Laurella solanacearum by adopting an M-SMSA solid culture medium, selecting a single colony of the Laurella solanacearum, transferring the single colony of the Laurella solanacearum into an NA liquid culture medium, and performing shake cultivation at 30 ℃ and 170rpm to obtain a ralstonia solanacearum bacterial liquid in a logarithmic phase.

The formula of the culture medium is as follows:

NA liquid medium: 10g of glucose, 5g of peptone, 3g of beef extract, 0.5g of yeast powder and 1000mL of deionized water, adjusting the pH value to 7.2-7.4, and carrying out autoclaving at 115 ℃ for 30 min.

M-SMSA solid medium (semi-selective solid medium for Ralstonia solanacearum): 50mg of TTC, 5mg of crystal violet, 50mg of polymyxin, 25mg of bacitracin, 5mg of chloramphenicol, 50mg of cycloheximide and 5mg of penicillin were added to 1000mL of NA solid medium.

Another objective of the invention is to provide a method for preventing and controlling tobacco soil-borne bacterial wilt by using the lytic bacteriophage FQ44, and a packageComprises the following steps: after the tobacco seedlings are transplanted for 7-30 days, adding a phage bacterium agent into the soil at the roots of the tobacco by a root irrigation method, wherein the final titer of the phage is 1 multiplied by 10⁷～1×10⁸PFU/g soil.

The invention has the beneficial effects that:

the method adopts a root irrigation method to apply the phage into the soil to inhibit the tobacco soil-borne bacterial wilt, the phage can only infect the ralstonia solanacearum, and the disturbance to the indigenous microbial community in the soil is reduced while the soil-borne bacterial wilt is prevented and controlled. Indoor related experiments show that the bacteriophage FQ44 belongs to Myocapnoraceae, can effectively inhibit the growth of part of ralstonia solanacearum, and has the best effect when applied according to the optimal multiplicity of infection (MOI 1-10); the field experiment effect also shows that the compound enzyme can effectively inhibit the occurrence of bacterial wilt and has wide prospect.

Drawings

FIG. 1 shows the plaque morphology of the bacteriophage FQ 44.

FIG. 2 shows the morphology of the bacteriophage FQ44 under a transmission electron microscope.

FIG. 3 is a one-step growth curve of bacteriophage FQ 44.

FIG. 4 shows the bacteriostatic effect of the phage FQ44 on the bacterial strain YNYC-23 of the host Ralstonia solanacearum.

FIG. 5 shows the host range of the phage FQ 44.

FIG. 6 shows the relationship between 28 strains of Ralstonia solanacearum.

FIG. 7 shows the effect of the bacteriophage FQ44 in field on inhibiting tobacco bacterial wilt.

Biological preservation information:

phage FQ44, classified and named as Laurella (Ralstonia solanacearum phase), was deposited at China Center for Type Culture Collection (CCTCC) at 29 months 9 and 2020, and the deposition unit address: the phage preservation number is CCTCC NO: m2020560.

The host bacterium YNYC-23, classified and named as Laurella solanacearum, was deposited in China center for type culture Collection at 29 months 9 and 2020, and the deposition unit address: the preservation number of the strains is CCTCC NO: m2020559.

Detailed Description

The technical solution of the present invention will be further explained with reference to the specific embodiments.

Example 1

Isolation of Ralstonia solanacearum

1g of tobacco plant rhizosphere soil sample collected from a bacterial wilt disease attack area in Yunnan province is taken and put into a sterilized 50mL triangular flask containing 9mL deionized water (water-soil ratio is 1: 9). And (3) putting the conical flask into a shaking table with the temperature of 30 ℃ and the rpm of 150-180 to cultivate for 30-60 min to obtain soil suspension. Taking a soil suspension for gradient dilution, and specifically comprising the following steps: taking 100 mu L of soil suspension, adding the soil suspension into a centrifuge tube containing 900 mu L of sterile water, and vortexing; a dilution gradient of 10 is obtained^-1The step is repeated to carry out gradient dilution to obtain a dilution gradient of 10^-2、10^-3、10^-4The soil suspension of (1). And taking 100 mu L of each dilution gradient soil suspension, and coating by using a ralstonia solanacearum semi-selective M-SMSA solid culture medium. Each dilution gradient was set to 3 replicates. The plate was placed upside down in an incubator at 30 ℃ for 48 hours. And selecting a single colony which is white at the edge, pink at the center and has fluidity, streaking the single colony on an M-SMSA culture medium, and culturing the single colony for 48 hours in a constant temperature incubator at the temperature of 30 ℃. This was repeated two to three times until a purified single strain was obtained. And selecting the purified single colony for 16S rRNA sequencing identification, determining the bacterial strain to be ralstonia solanacearum, and naming the bacterial strain to be YNYC-23. Selecting a single colony of ralstonia solanacearum, inoculating the single colony to an NA liquid culture medium, culturing the single colony in a shaking table at 30 ℃ and 150-180 rpm for 24 hours, adding equal volume of 30% glycerol, uniformly mixing, and storing in a refrigerator at-80 ℃.

YNYC-23, classified and named as Laurella solanacearum, was deposited in China center for type culture Collection at 29.9.2020, and the deposition unit address: the preservation number of the strains is CCTCC NO: m2020559.

Isolation of phages

A tobacco plant rhizosphere soil sample is collected in a bacterial wilt disease incidence area in Yunnan province, and a bacterial wilt special phage with high-efficiency cracking capacity is screened out by a double-layer agar plate method. The specific embodiment is as follows:

the ralstonia solanacearum YNYC-23 preserved by glycerol is streaked and activated on an M-SMSA solid culture medium, and is cultured in a constant temperature incubator at 30 ℃ for 48 hours. And (3) selecting a single colony on the plate, transferring the single colony into an NA liquid culture medium, and culturing for 24 hours in a shaking table at the temperature of 30 ℃ and the rpm of 170 to obtain bacterial liquid of ralstonia solanacearum YNYC-23 in logarithmic growth phase for later use.

Weighing 1g of collected fresh soil sample into a sterilized triangular flask containing glass beads, adding 9mL of sterile water, mixing uniformly, and placing in a shaking table at 30 ℃ and 170rpm for shake culture for 1 h. Transferring the enriched soil suspension into a sterilized 2mL centrifuge tube, centrifuging at 13000rpm for 3min, taking supernatant, and filtering (removing bacteria) through a 0.22-micron filter membrane to obtain filtrate, namely phage stock solution. Mixing 250 μ L bacterial liquid of Ralstonia solanacearum YNYC-23 in logarithmic growth phase with 25mL NA semisolid culture medium at proper temperature, immediately pouring into solidified NA solid culture medium to obtain double-layer plate containing Ralstonia solanacearum^[3]After the culture medium is solidified, 10 mu L of gradient dilution (10) is respectively spotted on different areas of the plate^-1，10^-2，10^-3，10^-4，10^-5，10^-6，10^-7，10^-8) The phage stock solution is cultured for 24 to 48 hours at the temperature of 30 ℃ and the existence of the plaques is observed.

When the plaques appear, selecting a single plaque with the maximum dilution, inoculating the single plaque into a bacterial solution of ralstonia solanacearum YNYC-23, carrying out shake culture at 170rpm for 24h at the temperature of 30 ℃ to proliferate the phage, carrying out high-speed centrifugation at 13000rpm for 3min on the suspension after the proliferation culture, taking the supernatant, filtering the supernatant through a 0.22 mu m filter membrane, and carrying out operation on the obtained filtrate by the double-layer agar plate method. And repeating for 2-3 times to obtain a relatively pure phage, wherein the phage obtained by purification exists in a phage suspension form, and the obtained new phage is numbered and is marked as phage FQ 44.

The formula of the culture medium is as follows:

NA semisolid medium: 10g of agar powder was added to 1L of NA liquid medium.

NA solid medium: 25g of agar powder was added to 1L of NA liquid medium.

M-SMSA solid medium: 50mg of TTC, 5mg of crystal violet, 50mg of polymyxin, 25mg of bacitracin, 5mg of chloramphenicol, 50mg of cycloheximide and 5mg of penicillin were added to 1000mL of NA solid medium.

Measurement of plaque diameter

Taking the purified phage suspension, and diluting in gradient (generally to 10)^-8) Then, the solution was dropped (10 to 20. mu.L) onto the corresponding positions of the NA semisolid culture medium plate containing Ralstonia solani (double-layer plate prepared by the above method) (in FIG. 1, 2, and 3 … … 8 represent dilution 10, respectively^-1、10^-2、10^-3……10^-8) And culturing at 30 ℃ for 24 h. Plaque formation was observed after 24h and the diameter of individual plaques was measured with a vernier caliper.

The diameters of individual plaques of phage FQ44 are shown in Table 1 and FIG. 1.

TABLE 1 plaque diameter

Example 2

Observation by phage electron microscope

1. Selecting the phage FQ44 to be observed and the corresponding host ralstonia solanacearum YNYC-23.

2. A double-layer plate of Ralstonia solanacearum was prepared according to the method of example 1, 150. mu.L of phage suspension was dropped on the plate, and the plate was incubated in a 30 ℃ incubator for 12-24 hours until plaques appeared.

3. Adding a small amount of sterile ddH dropwise at the spot where the plaque appears₂O (500-1000 mu L), standing for about 1h, and gently shaking for several times to allow the phage to float from the agarOn the water surface.

4. Taking a proper amount of phage suspension, carrying out negative dyeing by using 2% phosphotungstic acid dye liquor, taking out the phage suspension from the dye liquor after dyeing for about 90s, and naturally drying at room temperature.

5. The morphology of the phage was observed using a transmission electron microscope.

As seen in FIG. 2, the phage FQ44 had a regular hexahedral structure at the head, with a sheath, tail tube, basal disc and tail fiber. By comparison with the classification standard of the International Committee for the Classification of viruses^[4]The phage belongs to the order of tailed phages, family myocauda.

Example 3

Phage optimal multiplicity of infection assay

Multiplicity of infection (MOI) refers to the ratio of the number of phage to host bacteria before they are adsorbed and infect the host bacteria. The optimal multiplicity of infection is the MOI that achieves the highest progeny phage yield.

Bacterial suspension of Ralstonia solanacearum YNYC-23 in logarithmic growth phase obtained by the method described in reference example 1, and bacterial suspension OD of Ralstonia solanacearum was treated with sterile water₆₀₀The value was adjusted to 0.5. Dropwise adding the phage suspension obtained by purification in the example 1 into an NA liquid culture medium containing ralstonia solanacearum bacterial liquid, and carrying out shaking culture at 30 ℃ and 170rpm for 12 h; centrifuging the culture solution at 13000rpm at normal temperature for 3min, collecting supernatant, and filtering with 0.22 μm filter membrane to obtain bacteriophage suspension; adjusting phage suspension titer to 1 × 10 with sterile water⁸PFU/mL to obtain the phage inoculum.

Collecting bacterial solution (OD) of Ralstonia solanacearum₆₀₀0.5) adding into 48-well plate, 10 μ L per well, adding phage bacteria 10 μ L (each infection complex number is tested three times in parallel) according to MOI of 0.001, 0.01, 0.1, 1, 10, 100, shaking and mixing the mixed solution, placing into shaking table at 30 deg.C and 170rpm, shaking and culturing for 12h, sucking the co-culture suspension in 48-well plate into sterilized 2mL centrifuge tube, centrifuging at 13000rpm for 3min, collecting supernatant, filtering with 0.22 μm filter membrane to obtain phage suspension, diluting the phage suspension in gradient, and using double-layer agar plate method^[3]The titer is determined and recorded, and the optimal complex infection number can be obtained.

As shown in Table 2, the coculture solution having the multiplicity of infection of 0.001 and 0.01 was diluted in a gradient and dropped onto a double-layer plate containing Ralstonia solanacearum, and cultured for 24 hours, and no plaque was found. The highest phage titer was obtained at a multiplicity of infection of 1 and 10, and it was concluded that the optimal multiplicity of infection for phage FQ44 and YNYC-23 was between 1 and 10.

TABLE 2 optimal multiplicity of infection for bacteriophage FQ44

Example 4

Determination of one-step growth curves of bacteriophages

Culturing Ralstonia solanacearum YNYC-23 to logarithmic growth period, and adjusting bacterial liquid OD₆₀₀The value was 0.5, 10 μ L of the bacterial suspension was added to 48-well plates in sequence, and phage inoculum (example 3, titer 1 × 10) was added according to the optimal multiplicity of infection (MOI ═ 10)⁸PFU/mL)10 μ L, shaking culture at 30 deg.C and 170rpm, sampling every hour, determining for 12 hr, repeating for 3 times, taking out, centrifuging at 13000rpm for 3min, filtering to obtain supernatant, and performing double-layer agar plate method^[3]And (4) measuring the titer, and drawing a one-step growth curve of the phage by taking the titer logarithmic value of the phage as the ordinate and the culture time as the abscissa.

The phenomenon that the titer of the phage does not increase with time after the phage infects the host bacteria is called latency, and the outbreak period is the trend that the phage rapidly increases with time.

As shown in FIG. 3 and Table 3, the incubation period of the phage FQ44 is 1h, the outbreak period lasts for 9h, the number of the phage increases the fastest in the 5 th to 8 th hours, then the growth speed of the phage slows down, and the phage does not change basically after 10 h.

TABLE 3 one-step growth curves for bacteriophage FQ44

Example 5

Detection of phage infectivity

Adjusting the titer of the phage inoculum of example 3 to 10⁹PFU/mL; OD of bacterial liquid of Ralstonia solanacearum YNYC-23 with reference to the method of example 3₆₀₀The adjustment was 0.5.

The capability of the phage FQ44 to infect the ralstonia solanacearum YNYC-23 is detected in a 96-well plate, and 3 treatments are set in total: (1) and (3) co-culture group: adding 180 mu LNA liquid culture medium, 10 mu L of ralstonia solanacearum liquid and 10 mu L of phage bacterium agent into each hole; (2) single culture group: adding 190 μ LNA liquid culture medium and 10 μ L Ralstonia solanacearum liquid into each well; (3) blank control: add 200. mu. LNA liquid medium per well (to see if the medium is contaminated). Each process set 3 replicates. Respectively measuring OD of 0h, 8h, 16h and 24h by using an enzyme-labeling instrument₆₀₀Numerical values.

The results are shown in FIG. 4 and Table 4. The inhibition effect of the phage in the first 8h is not obvious, the ralstonia solanacearum co-cultured with the phage after 8h is obviously inhibited, the biomass of the ralstonia solanacearum is not increased any more, the inhibition effect of the phage after 16h is obvious, and the biomass of the ralstonia solanacearum in the co-culture solution is reduced. In general, phage FQ44 significantly inhibited the growth of the host Ralstonia solanacearum YNYC-23.

TABLE 4 inhibitory Effect of bacteriophage FQ44 on host tobacco Ralstonia solanacearum YNYC-23

Example 6

Host Range of phage FQ44

Ralstonia solanacearum has a high diversity^[2]And the anti-phage mechanism of different ralstonia solanacearum^[5]Different. Therefore, 28 strains of ralstonia solanacearum derived from tobacco rhizosphere soil of Yunnan province, Guizhou province, and Guangxi province were selected to determine the host range of the phage FQ 44. The strains of WSD1-1, WSD1-3, WSD5-3, ysqk-1-14 and YNYC are from ralstonia solanacearum in different areas of Yunnan province. The ralstonia solanacearum GZ202 is from tobacco rhizosphere soil in Guizhou, and the ralstonia solanacearum tb06, tb08 and tb23 are from tobacco rhizosphere soil in Guangxi province.

Referring to example 3, the phage suspension and the bacterial solution of Ralstonia solanacearum were obtained, and the phage preparation was adjustedTiter to 10⁸PFU/mL, OD of bacterial liquid of each strain of Ralstonia solanacearum₆₀₀The adjustment was 0.5. A small amount of phage suspension is dripped into a flat plate (130mm x 130mm) containing NA solid culture medium, so that the phage suspension vertically flows from one side of the flat plate to the other side under the action of gravity, a liquid transfer gun is used for sucking away excessive phage suspension, after the phage suspension is naturally dried, 12 branched inoculating needles are used for dipping bacterial liquid of 12 ralstonia solanacearum, and the part through which the phage suspension flows is forcibly and uniformly scratched. The experiment was performed separately with 28. ang. ralstonia solanacearum strains and the phage FQ44 (each ralstonia solanacearum strain was considered as one treatment), with three replicates per treatment set up. After culturing at 30 ℃ for 24h, the cyanobacterium endemicum passed by the phage suspension is hindered from growing, i.e., the phage FQ44 can infect the cyanobacterium.

As shown in FIG. 5, the bacteriophage FQ44 can infect a significant portion of Ralstonia solanacearum from Yunnan and cannot infect a portion of Ralstonia solanacearum from Guizhou and Guangxi. As shown in FIG. 6, even from the same province, there was a large difference between these P.nicotianae, and the P.nicotianae which could be infected with the phage FQ44 was mainly concentrated in 3 sequence variants. In conclusion, the bacteriophage FQ44 can infect different ralstonia solanacearum, mainly ralstonia solanacearum in Yunnan, and the sequence variants thereof are mainly 11, 16 and 44.

Example 7

Tobacco field tests were conducted in the black field of Shanpu, Yunnan province.

Reference example 3 phage suspension was prepared, and phage titer was adjusted to 10 with sterile water⁸PFU/mL to obtain the phage inoculum.

2 treatments are set: (1) phage group: after the tobacco is transplanted to the field for 30 days, uniformly irrigating 200mL phage inoculum around the root of each tobacco plant, wherein the final titer of phage is 1 × 10⁸PFU/g soil, each tobacco is calculated according to 200g rhizosphere soil; (2) control group: after the tobacco was transplanted to the field for 30 days, 200mL of sterile water was poured around the roots of each tobacco plant. Each process is done in 3 repetitions (cell). Other management measures are consistent with the routine operation of local farmers. And counting the number of the plants with the disease after the tobacco has the disease symptoms of the bacterial wilt until the disease is stable.

The formula for calculating the incidence is as follows:

incidence (%). the number of diseased plants per total number of plants in the plot x 100%.

The results are shown in table 5 and fig. 7. After the tobacco is irrigated with the phage, the incidence rate of bacterial wilt is remarkably reduced (P < 0.05).

TABLE 5 Effect of bacteriophage FQ44 on field inhibition of tobacco bacterial wilt

Note: different letters represent significant differences in P <0.05 levels.

Reference documents:

[1].Abedon ST.Phage therapy:eco-physiological pharmacology.Scientifica(Cairo)2014；2014:581639.

[2].Jiang G,Wei Z,Xu J,et al.Bacterial Wilt in China:History,Current Status,and Future Perspectives.Front Plant Sci 2017；8:1549.

[3].Breitbart M,Rohwer F.Here a virus,there a virus,everywhere the same virusTrends Microbiol 2005；13:278-84.

[4].Ofir G,Sorek R.Contemporary Phage Biology:From Classic Models to New Insights.Cell2018；172:1260-70.

[5].Labrie SJ,Samson JE,Moineau S.Bacteriophage resistance mechanisms.Nat Rev Microbiol 2010；8:317-27.

Claims

1. a lytic bacteriophage FQ44, classified and named as Laurella sp (Ralstonia solanacearum phage), is preserved in China center for type culture Collection in 29 months 9 and 2020, and has a preservation number of CCTCC NO: m2020560.

2. The use of the lytic bacteriophage FQ44 of claim 1 for preventing and controlling soil-borne bacterial wilt of tobacco.

3. Use according to claim 2, characterized in that the phages are applied to the soil by the root irrigation method.

4. Use according to claim 2 or 3, characterized in that the final titer of the phages is 1X 10⁷～1×10⁸PFU/g soil.

5. A phage preparation for preventing and controlling tobacco soil-borne bacterial wilt, characterized in that the phage preparation comprises the lytic phage FQ44 of claim 1, and the titer of the phage preparation is 1 x 10⁸～1×10⁹PFU/mL。

6. A method for preventing and controlling soil-borne bacterial wilt of tobacco using lytic bacteriophage of claim 1, comprising: after the tobacco seedlings are transplanted for 7-30 days, adding a phage bacterium agent into the soil by a root irrigation method, wherein the final titer of the phage is 1 multiplied by 10⁷～1×10⁸PFU/g soil.

7. The method of claim 6, wherein the phage inoculum is prepared by: culturing with NA liquid culture medium to obtain Ralstonia solanacearum liquid in logarithmic growth phase, and culturing Ralstonia solanacearum liquid OD with sterile water₆₀₀Adjusting the value to 0.45-0.55; the titer of the phage inoculum is adjusted to 1 multiplied by 10 by sterile water⁸～1×10⁹PFU/mL, placed at OD according to optimal multiplicity of infection₆₀₀Performing shaking culture at 30 ℃ and 150-180 rpm for 9-12 h in a bacterial solution of ralstonia solanacearum with the value of 0.45-0.55, centrifuging at 12000-14000 rpm, taking supernatant, and filtering with a 0.22 mu m filter membrane; regulation of phage titer to 1X 10 with sterile water⁸～1×10⁹PFU/mL to obtain phage preparation.

8. The method of claim 7, wherein the optimal multiplicity of infection is 1 to 10.