Use of streptomyces verticillus phage φs102

Abstract

The present application relates to a long tail wide host spectrum streptomyces strong lytic bacteriophage and its application in the prevention and treatment of potato scab medicine. The long tail wide host spectrum streptomyces strong lytic bacteriophage belongs to the long tail bacteriophage family, has strong lysis to wide host spectrum streptomyces, and has high titer. The long tail wide host spectrum streptomyces strong lytic bacteriophage is used for preventing and treating potato scab, has good safety, good effect and no pollution. The experimental results of the bacteriophage for preventing and treating potato scab show that the average coverage rate of scab lesions of the control group is about 28.5%, compared with the bacteriophage treatment group, the lesion coverage rate of the tuber offspring is only 3.5%, which is significantly reduced (p<0.05). Through bacteriophage treatment, the damage type and damage number of each tuber are also significantly reduced (p<0.05). There is no significant difference in tuber weight, size or number between the bacteriophage treatment and the untreated tubers.

Description

[0001] This invention is a divisional application of “A long-tailed, broad-host-spectrum Streptomyces with strong lytic activity and its application in drugs for the prevention and treatment of potato scab (Application No. 2021106907012, Application Date: June 22, 2021)”. Technical Field

[0002] This invention belongs to the field of microbial technology, specifically relating to the application of a Streptococcus scabra bacteriophage φS102. Background Technology

[0003] Potato scab is caused by the pathogen Streptomyces (… S.scabies, S.galilaeus, S.bobili Bacterial diseases of potatoes (such as those caused by various factors) damage potato tubers, resulting in nearly circular to irregular corky, scab-like light brown lesions or patches on the tuber surface, with a rough texture to the touch. Lesions are usually limited to the cortex, but sometimes runners can also be affected, reducing the quality and yield of affected tubers, making them less resistant to storage, and significantly lowering their marketable quality due to their unsightly appearance. Although comprehensive control measures such as rational layout, promotion of disease-resistant varieties, seed potato treatment, knife disinfection, establishment of seed potato fields, inter-row cultivation and hilling, and pesticide protection can effectively control bacterial diseases of potatoes, the multiple stages of treatment increase labor intensity, leading to high industry costs. Furthermore, the extensive use of pesticides has resulted in the emergence of super-resistant bacteria, serious environmental pollution, and excessive pesticide residues in commercial potatoes. Therefore, there is an urgent need to develop efficient, safe, and environmentally friendly new technologies and agents for the control of bacterial diseases of potatoes.

[0004] Bacteriophages are a general term for viruses that infect microorganisms such as bacteria, fungi, algae, actinomycetes, or spirochetes, and their numbers exceed those of bacteria by about ten times. Currently, bacteriophages hold promise as antimicrobial agents to replace antibiotics and pesticides in addressing the increasingly serious problem of antibiotic resistance in pathogenic bacteria. Simultaneously, due to their high specificity, ease of isolation and proliferation, low genotoxicity, and tolerance to food processing environments, bacteriophages are also expected to serve as ideal biological preservatives or detection methods in various fields. Bacteriophages proliferate rapidly, and once they colonize a pathogenic host, there is no need for further replenishment. When pathogens are infected with a specific bacteriophage, they may become incurable, much like a malignant tumor, thus eradicating diseases that were not originally found in a particular area and maintaining the original local ecosystem. Compared to using pesticides to control the spread of diseases, bacteriophages represent a better biological control method in terms of both environmental friendliness and reduced production costs. Summary of the Invention

[0005] To address the aforementioned problems in existing technologies, this invention provides a safe, effective, and pollution-free bacteriophage and its application in drugs for the prevention and treatment of potato scab. The bacteriophage belongs to the family Longtailophageaceae and exhibits strong lytic activity against broad-spectrum Streptomyces, demonstrating high potency.

[0006] The technical solution adopted in this invention is as follows: Streptomyces scabii bacteriophage ( Streptomyces The strain phage φS102 was deposited on April 15, 2021, at the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China, 430072, China), with accession number CCTCC NO:M 2021379.

[0007] The bacteriophage has a polyhedral head structure and a non-contractile tail. The head diameter is about 64±7 nm and the tail length is about 196±8 nm. The bacteriophage belongs to the family Longtail Phageidae.

[0008] The bacteriophage is active in the pH range of 2-10, and its activity is stable at 30℃-80℃, but it is inactivated above 80℃.

[0009] The incubation period of the bacteriophage is 20 minutes, and the outbreak period is 60 minutes.

[0010] The phage had a titer of 1.47 × 10⁻⁶ at an MOI of 0.01. 7 .

[0011] The method for isolating and purifying the highly lytic phage of Streptomyces with a long tail and broad host spectrum is as follows: (1) Sample pretreatment: After scraping off the skin of the potato with diseased spots and grinding it, distilled water was added. The mixture was then cultured at 30°C and 170 rpm for 6-8 hours with shaking. The supernatant was then collected, centrifuged at 4000 rpm for 10 minutes, and filtered to obtain a sample solution containing bacteriophages. (2) Preparing the host bacteria: Single colonies of Streptomyces that grew well on the plate were inoculated into Gao's liquid medium and cultured at 170 r / min and 30 ℃ until the early logarithmic growth stage, and then set aside for later use. (3) Proliferation and isolation of bacteriophages Take 10 mL of the sample solution containing bacteriophage and add it to 30 mL of Gao's No. 1 liquid culture medium. Add 1 mL of host bacteria suspension in the early logarithmic growth phase and a certain amount of calcium chloride. Mix thoroughly to make the final concentration of calcium chloride in the system 10 mmol / L. Incubate overnight at 30℃ and 170 r / min with shaking. Centrifuge the culture solution at 4℃ and 10000 r / min for 10 min. Collect the supernatant and filter to obtain the bacteriophage enrichment solution. In a sterile laminar flow hood, 100 µL of host bacterial culture was evenly spread onto a Gao's solid medium plate and allowed to stand for 15 min. Then, 100 µL of phage enrichment solution diluted 10-1000 times with SM solution was added to the plate with the host bacterial culture and spread evenly. After standing for 15 min, the plate was inverted and incubated overnight to form phage plaques. (4) Purification of bacteriophages Using a sterile toothpick, select a single, uniformly sized, and translucent phage plaque and place it in 1 mL of SM solution. Incubate at 30°C with shaking at 170 rpm, then filter. Dilute the filtrate 10 times with sterile SM solution. 3 -10 6 After multiplying, continue plating and purify according to the above steps. Repeat this process multiple times to obtain purified phage plaques. Select uniform, clear, and single phage plaques and place them in SM solution. After shaking culture and filtration, a purified phage solution is obtained.

[0012] In step (2), the Streptomyces is S.scabies, S.galilaeus, S.bobili any one of them 。

[0013] In step (4), the phage solution is stored at 4°C and revived every six months.

[0014] In step (4), the phage solution is mixed with 60% glycerol in a 1:2 ratio and then placed in a cryovial for long-term storage at -80°C, with periodic thawing.

[0015] This invention provides the application of Streptomyces scab phage φS102, specifically including the application of the aforementioned Streptomyces long-tailed, broad-host-spectrum, highly lytic phage in the preparation of drugs for the prevention and control of potato scab disease, and also includes a method for the prevention and control of scab disease using Streptomyces scab phage φS102.

[0016] The present invention has the following specific beneficial effects: The *Streptomyces scabica* phage φS102 described in this invention belongs to the family Longtail Phagesaceae and exhibits strong lytic activity against broad-spectrum *Streptomyces*, with high titer. The *Streptomyces scabica* phage φS102 described in this invention is safe, effective, and pollution-free for the control of potato scab disease.

[0017] Experimental results on the control of potato scab disease using bacteriophages showed that the average scab lesion coverage rate in the control group was approximately 28.5%, while the lesion coverage rate in the tuber progeny of the bacteriophage-treated group was only 3.5%, a significant decrease (p<0.05). Furthermore, bacteriophage treatment significantly reduced the type and number of lesions per tuber (p<0.05). There were no significant differences in tuber weight, size, or number between bacteriophage-treated and untreated tubers. Attached Figure Description

[0018] 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 some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a morphological observation image of the bacteriophage φS102 described in this invention; Figure 2 This refers to the pH stability of the bacteriophage φS102 described in this invention; Figure 3 The temperature stability of the bacteriophage φS102 described in this invention; Figure 4 This is a one-step growth curve of the bacteriophage φS102 described in this invention. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some examples of this invention, and not all examples. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0021] Streptomyces scabii bacteriophage ( Streptomyces The strain phage φS102 was deposited on April 15, 2021, at the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China, 430072, China), with accession number CCTCC NO:M 2021379.

[0022] Example 1 The method for isolating and purifying Streptomyces scabii bacteriophage φS102 described in this invention is as follows: (1) Sample pretreatment: After scraping off the skin of the diseased potato and grinding it, add 50 mL of distilled water and place it in a 250 mL Erlenmeyer flask. Incubate at 30 °C and 170 rpm for 6-8 h with shaking. Take the supernatant, centrifuge the supernatant at 4000 rpm for 10 min and filter it through a 0.22 µm microporous filter to obtain a sample solution containing bacteriophages. (2) Preparing the host bacteria: A single colony of Streptomyces that grew well on the plate was inoculated into 100 mL of Gao's liquid medium and cultured at 170 r / min and 30 ℃ until the early logarithmic growth stage, and then set aside for later use. (3) Proliferation and isolation of bacteriophages Take 10 mL of the sample solution containing bacteriophage and add it to 30 mL of Gao's No. 1 liquid culture medium. Add 1 mL of host bacteria suspension in the early logarithmic growth phase and a certain amount of calcium chloride. Mix thoroughly to make the final concentration of calcium chloride in the system 10 mmol / L. Incubate overnight at 30℃ and 170 r / min with shaking. Centrifuge the culture solution at 4℃ and 10000 r / min for 10 min. Collect the supernatant and filter it through a 0.22 µm microporous filter to obtain the bacteriophage enrichment solution. In a sterile laminar flow hood, 100 µL of host bacterial culture was evenly spread onto a Gao's solid medium plate and allowed to stand for 15 min. Then, 100 µL of phage enrichment solution diluted 10-1000 times with SM solution was added to the plate with the host bacterial culture and spread evenly. After standing for 15 min, the plate was inverted and incubated overnight to form phage plaques. (4) Purification of bacteriophages Using a sterile toothpick, select a single, uniformly sized, and translucent phage plaque and place it in 1 mL of SM solution. Incubate at 30°C with shaking at 170 rpm, then filter. Dilute the filtrate 10 times with sterile SM solution. 3 -10 6 After multiplying, continue plating and purify according to the above steps. Repeat this process multiple times to obtain purified phage plaques. Select uniform, clear, and single phage plaques and place them in SM solution. After shaking culture and filtration, a purified phage solution is obtained. (5) Preservation of bacteriophages Phage solutions can be stored long-term at 4°C, requiring resuscitation every six months. Alternatively, a phage solution can be mixed with 60% glycerol at a 1:2 ratio, placed in cryovials, and stored at -80°C for long-term storage, requiring periodic resuscitation.

[0023] The bacteriophage was named φS102. It has a polyhedral head structure and a non-contractile tail. The head diameter is approximately 64±7 nm, and the tail length is approximately 196±8 nm. On a double-layer agar plate, the bacteriophage forms uniformly shaped and translucent plaques with no halo, clear and regular edges, and a diameter of approximately 3 mm. This bacteriophage belongs to the family Longtail Phageidae (φS102). Figure 1 ).

[0024] Example 2 Titer and optimal multiple of infection assay of Streptomyces scabies phage φS102 1. The titer of Streptomyces cuspidatum bacteriophage was determined: Phage titer refers to the number of infectious phages per milliliter of sample. The formula for calculating phage titer is as follows: Phage titer (PFU / mL) = number of phage plaques × dilution factor / phage volume.

[0025] Take 100µL of host bacterial culture, spread it on a plate, and let it stand until dry. Then, take a plate diluted 10 μL with sterile SM solution. 3 -10 6 Spread 100µL of the phage solution onto a plate covered with host bacteria, incubate upside down at 30°C, and observe the number of phage plaques on the plate. Repeat the experiment 3 times, with 3 replicates each time.

[0026] 2. Determination of the optimal multiple of infection for bacteriophages The multiple of infection (MOI) is the ratio of the number of phages added at the time of initial infection to the number of host bacteria. 100 μL of host bacteria and 100 μL of phage were added to Gao's liquid medium at MOIs of 100, 10, 1, 0.1, 0.01, 0.001, and 0.0001, respectively. The medium was incubated at 30°C with shaking at 170 rpm and filtered through a 0.22 μm filter to obtain purified phage solution. The phage solution was serially diluted from 10⁻¹ to 10⁻⁹, and the phage titer was determined using the plate spread method. The MOI with the highest titer was the optimal MOI. The experiment was repeated three times, with three replicates per experiment.

[0027] The details are shown in Table 1.

[0028] Table 1 - Optimal Multiplicity of Infection for Bacteriophage φS102

[0029] The results showed that the titer of bacteriophage φS102 could reach 1.47 × 10⁻⁶ at an MOI of 0.01. 7 .

[0030] Example 3 The host spectrum of the bacteriophage (named φS102) was determined using the bacteriophage spot method: Thirty-four isolated Streptomyces strains capable of causing scab disease were streaked onto agar plates, and single colonies with good morphology were selected for culture. 100 µl of the culture, cultured to the early logarithmic growth stage, was spread onto an agar plate. After the culture dried, 10 µl of phage stock solution was pipetted onto the plate, ensuring proper spacing between each drop. The plate was incubated at 30°C after the solution had completely dried. The plate was then inverted and incubated the next day. The presence of phage plaques on the bacterial growth was observed the following day; the appearance of phage plaques indicated that the phage could lyse the bacteria. Details are shown in Table 2 below.

[0031] Table 2 - Host range of bacteriophage φS102

[0032] The results showed that the bacteriophage φS102... S.scabies, S.galilaeus, S.bobili All of them exhibited lytic activity. The bacteriophage φS102 showed lytic activity against 14 of the 34 Streptomyces strains that caused potato scab collected.

[0033] Example 4 The effects of temperature and pH on phage stability: The potency reaches 10 8 One mL of phage solution was placed in a sterilized EP tube and placed in a water bath at different temperatures. After 1 hour, the tube was immediately removed and placed in an ice bath to cool. The phage titer was then determined using the plate spread method at 30℃, 40℃, 50℃, 60℃, 70℃, 80℃, 90℃, and 100℃. The experiment was repeated three times, with three replicates each time.

[0034] Prepare SM solution with a pH of 1-12, transfer it to sterile EP tubes, and incubate at 30°C for 30 minutes until temperature equilibration. This will bring the potency to 10. 8 Each time, 100 μL of phage fluid was added to 900 μL of SM solution at different pH values. The solutions were then incubated in a water bath at 30°C for 1 hour. After incubation, the EP tubes were removed, and the titer of the phage samples at different pH values ​​was determined using the plate spread method. The experiment was repeated three times, with three replicates.

[0035] The results showed that the bacteriophage φS102 was active in the pH range of 2-10, and its activity was stable. Figure 2 The bacteriophage activity is stable between 30℃ and 80℃, but it is inactivated above 80℃. Figure 3 ).

[0036] Example 5 One-step growth curve determination of bacteriophages: Phages and host bacteria were mixed at the optimal multiplicity of infection (MOI), and the mixture was shaken at 30°C and 170 rpm for 15 min to adsorb the phages. The mixture was then centrifuged at 10,000 rpm for 10 min, the supernatant was discarded, and the phages were resuspended in fresh liquid culture medium. The mixture was washed 2-3 times to remove any unadsorbed phages. 15 mL of Gao's liquid medium was added and thoroughly mixed. The mixture was then quickly placed in a shaking incubator and incubated at 30°C and 170 rpm. Starting from 0°C, samples were taken every 10 min at regular intervals. After aseptic filtration, the phage titer in the supernatant was determined using the plate spread method. A one-step growth curve was plotted, with phage titer on the ordinate and sampling time on the x-axis. The experiment was repeated three times, with three replicates per run. The results are as follows: Figure 4 As shown, the incubation period of bacteriophage φS102 is 20 min, and the outbreak period is 60 min.

[0037] Example 6 Control of potato scab by Streptomyces scabii bacteriophage φS102: One hundred seed potatoes with a diameter of approximately 2 cm and suffering from scab disease were selected and randomly divided into two groups. One group was soaked in an immersion solution with a titer of 1×10⁻⁶. 7 Seed potatoes were soaked in bacteriophage suspension at 25°C for 24 hours. Another group underwent the same treatment, but the bacteriophage suspension was autoclaved at 121°C for 20 minutes before soaking. The soaked seed potatoes were planted at the same depth in polystyrene foam boxes, which allowed for water permeability. The soil inside the boxes was sterilized by steam at 60°C for 1 hour. Five seed potatoes were planted in each foam box, with 10 replicates per group. The foam boxes were placed in a greenhouse at 22°C ± 2°C. Watering was done daily for the first two weeks after planting, then reduced. Progeny tubers were harvested after 60 days. The severity of scab damage was assessed by counting the percentage of damaged surface area and damage type on each progeny tuber. The lesion surface area (%) was graded on a scale of 0-6 as follows: 0, no lesion; 1, ≤10%; 2, 11–20%; 3, 21–30%; 4, 31–40%; 5, 41–50%; 6, 50%. Damage types were graded on a scale of 0-4 as follows: 0, no damage; 1, superficial lesions; 2, slightly raised or sunken lesions; 3, sunken lesions; 4, deeply sunken lesions. The weight, size, and number of tubers of each plant were examined to determine differences between the two groups.

[0038] The results are shown in Table 3.

[0039] Table 3 - Control efficacy of Streptomyces scabii bacteriophage φS102 against potato scab in progeny.

[0040] Table 3 shows that the average lesion coverage rate of the control group was approximately 28.5%, while the lesion coverage rate of the tuber progeny in the phage-treated group (3.5%) was significantly lower (p<0.05). Furthermore, phage treatment significantly reduced the type of damage (scab type) and the number of damages (scab spots) per tuber (p<0.05). There were no significant differences in tuber weight, size, or number between phage-treated and untreated tubers. The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. Streptomyces scabii bacteriophage ( Streptomyces The application of phage)φS102 in Streptomyces lysus, characterized in that, The accession number for Streptomyces scabii bacteriophage φS102 is CCTCC NO:M 2021379; The bacteriophage φS102 is active in the pH range of 2-10 and its activity is stable at 30℃-80℃. The Streptomyces is any one of S. scabies, S. galilaeus, and S. bobili.

2. A treatment method for potato scab, characterized in that, The treatment method includes the step of applying Streptomyces scabica phage φS102 to potatoes, wherein the preservation number of Streptomyces scabica phage φS102 is CCTCC NO:M2021379.

3. The treatment method according to claim 2, characterized in that, The treatment method involves soaking seed potatoes in a suspension of Streptomyces scabii bacteriophage φS102 for 24 hours.

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