CN116355862A - Application of streptomyces scab phage phi S102 - Google Patents
Application of streptomyces scab phage phi S102 Download PDFInfo
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Abstract
The invention relates to a Streptomyces longtail broad-host spectrum strong lytic bacteriophage and application thereof in a medicine for preventing and treating potato scab. The invention relates to a Streptomyces longus and broad host spectrum strong lytic bacteriophage, belonging to longus bacteriophage family, which has strong lytic property to Streptomyces longus and broad host spectrum and high potency. The Streptomyces longtail broad-host spectrum strong-lytic phage is used for preventing and treating potato scab, and has the advantages of good safety, good effect and no pollution. Experimental results of the phage for preventing and treating potato scab show that: the control group had an average scab lesion coverage of around 28.5%, compared to only 3.5% for the tuber offspring of the phage-treated group, with a significant reduction (p < 0.05). The type of injury and the number of lesions per tuber was also significantly reduced by phage treatment (p < 0.05). There were no significant differences in tuber weight, size or number between phage treated and untreated tubers.
Description
The invention relates to a Streptomyces longtail broad-host spectrum strong lytic bacteriophage and application thereof in a medicine for preventing and treating potato scab (application number 2021106907012, application date: 2021, 06 and 22 days).
Technical Field
The invention belongs to the technical field of microorganisms, and in particular relates to a streptomyces scab bacteriophage
Is used in the application of (a).
Background
Potato scab is a plant disease caused by pathogenic streptomyces (s. Scices, s. Galilaeus, s. Bobili, etc.), which harms the potato tubers, resulting in the appearance of nearly circular to amorphous wood-bolting scab-like pale brown lesions or plaques on the tuber surface, with rough hand feel; usually, the disease spots are limited to the cortex, sometimes the stolon can be damaged, so that the quality and the yield of the damaged potato blocks are reduced, the damaged potato blocks are not storage-resistant, the appearance of the damaged potato blocks is elegant, and the commodity grade is greatly reduced. Although the bacterial diseases of the potatoes can be effectively controlled by implementing comprehensive prevention and treatment measures such as reasonable layout, popularization of disease-resistant varieties, potato seed treatment, cutter disinfection, establishment of potato seed fields, cultivation and hilling, pesticide spraying protection and the like, the labor intensity is increased by the treatment of a plurality of links, and the industrial cost is kept high; on the other hand, the use of a large amount of pesticides causes the occurrence of super-resistant bacteria, serious environmental pollution, excessive pesticide residues of commercial potatoes and other problems, so that in order to support the national potato staple food strategy and the healthy development of potato industry, development of a novel technology and a novel medicament for preventing and controlling potato bacterial diseases with high efficiency, safety and environmental protection are needed.
Phage is a general term for viruses that infect microorganisms such as bacteria, fungi, algae, actinomycetes, or spirochetes, and is about ten times more than bacteria in number. At present, phage is expected to be used as an antibacterial agent to replace antibiotics and pesticides so as to solve the problem of increasingly serious drug resistance of pathogenic bacteria; meanwhile, due to the characteristics of strong specificity, easy separation and increment, low genetic toxicity, food processing environment tolerance and the like, the phage is expected to be used as an ideal biological preservative or detection means in various fields. The phage proliferate rapidly and after colonizing the pathogenic host, no phage need to be replenished. After the pathogenic bacteria infect a certain corresponding bacteriophage, the disease can not be cured like malignant tumor, so that the corresponding disease which does not belong to a certain place originally can be eradicated, and the local original ecological environment is maintained. Compared with the use of pesticides to control the spread and spread of diseases, phage is a better biological control method, both from the environmental point of view and the reduction of production costs.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a safe, effective and pollution-free bacteriophage and application thereof in medicines for preventing and treating potato scab. The phage belongs to the family of long tail phage, has strong cracking property on streptomyces with wide host spectrum and has high titer.
The technical scheme adopted by the invention is as follows:
streptomyces scabus phage (Streptomyces phage)
The strain is preserved in China center for type culture Collection (address: university of Chinese Wuhan, post code 430072) at 4 months and 15 days of 2021, and the preservation number is CCTCC NO: M2021379.
The phage has a polyhedral head structure and a non-contractile tail, the head diameter is about 64+/-7 nm, the tail length is about 196+/-8 nm, and the phage belongs to the long-tailed phage family.
The phage has activity in the pH range of 2-10, the phage has stable activity at 30-80 ℃ and is inactivated at more than 80 ℃.
The incubation period of the phage was 20min and the burst period was 60min.
The phage had a titer of 1.47X 10 at MOI of 0.01 7 。
The separation and purification method of the Streptomyces longtail broad-host spectrum strong-lytic phage comprises the following specific steps:
(1) Sample pretreatment:
scraping the epidermis of the potato with the disease spots, grinding, adding distilled water, shaking and culturing at 30 ℃ and 170rmp for 6-8 hours, taking supernatant, centrifuging the supernatant at 4000r/min for 10 minutes, and filtering to obtain a sample liquid containing phage;
(2) Preparing a host bacterium:
inoculating single bacterial colony of streptomycete with good growth on a flat plate into a Gao's liquid culture medium, and culturing at 170r/min and 30 ℃ until logarithmic growth is early for later use;
(3) Proliferation isolation of phages
Adding 10mL of a sample solution containing phage into 30mL of a Gao's first liquid culture medium, adding 1mL of a host bacterium suspension in the early logarithmic phase growth phase, adding a certain amount of calcium chloride, fully and uniformly mixing to ensure that the final concentration of the calcium chloride in the system is 10mmol/L, performing shake culture at 30 ℃ and 170r/min overnight, obtaining a culture solution, centrifuging at 4 ℃ and 10000r/min for 10min, collecting supernatant, and filtering to obtain a phage enrichment solution;
sucking 100 mu L of host bacterial liquid in a sterile super clean bench, uniformly coating the host bacterial liquid on a Gao's solid culture medium flat plate, standing for 15min, then sucking 100 mu L of phage enrichment liquid diluted 10-1000 times by SM liquid on the flat plate coated with the host bacterial liquid, uniformly coating the phage enrichment liquid, standing for 15min, and then inversely culturing overnight to form plaque;
(4) Purification of phages
Picking with sterile toothpickPlacing single plaque with uniform size and transparent in 1mL SM solution, shake culturing at 30deg.C at 170r/min, filtering, and diluting filtrate with sterile SM solution for 10 3 -10 6 After doubling, continuing to plate, purifying according to the operation, and repeating for a plurality of times to obtain purified plaque;
and (3) picking uniformly transparent single plaque, placing the plaque in SM solution, and obtaining purified phage solution after shake culture and filtration.
In the step (2), the streptomycete is any one of S.scabies, S.galilaeus and S.bobili.
In the step (4), phage solution is stored at 4 ℃ and recovered every half year.
In the step (4), phage solution and 60% glycerol are mixed according to the proportion of 1:2, and then are put into a freezing tube for long-term storage at-80 ℃ and are periodically resuscitated.
The invention provides Streptomyces scab phage
The application of the streptomyces longifolius strong-lytic phage in preparing a medicine for preventing and treating potato scab and the application of the streptomyces scab phage->
A method for preventing and treating scab is provided.
The invention has the following advantages:
the streptomyces scab phage of the invention
Belongs to the family of long tail phage, has stronger cracking property to streptomyces with wide host spectrum and high potency. The streptomyces scab bacteriophage of the invention>
The composition is used for preventing and treating potato scab, and has good safety, good effect and no pollution.
Experimental results of phage control of potato scab show that: the control group had an average scab lesion coverage of around 28.5%, compared to only 3.5% for the tuber offspring of the phage-treated group, with a significant reduction (p < 0.05). Furthermore, the type of injury and the number of lesions per tuber was also significantly reduced by phage treatment (p < 0.05). There were no significant differences in tuber weight, size or number between phage treated and untreated tubers.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows a phage according to the invention
Morphology view of (a);
FIG. 2 shows a phage according to the invention
pH stability of (c);
FIG. 3 shows a phage according to the invention
Temperature stability of (2);
FIG. 4 shows a phage according to the invention
Is a one-step growth profile of (a).
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It will be apparent that the embodiments described are only some, but not all, embodiments of the invention. All other embodiments, based on the examples herein, which are within the scope of the invention as defined by the claims, will be within the scope of the invention as defined by the claims.
Streptomyces scabus phage (Streptomyces phage)
The strain is preserved in China center for type culture Collection (address: university of Chinese Wuhan, post code 430072) at 4 months and 15 days of 2021, and the preservation number is CCTCC NO: M2021379.
Example 1
The streptomyces scab phage of the invention
The separation and purification method of (2) is specifically as follows:
(1) Sample pretreatment:
scraping the epidermis part of the potato with the disease spots, grinding, adding 50mL of distilled water, placing in a 250mL conical flask, shaking and culturing at 30 ℃ and 170rmp for 6-8 hours, taking supernatant, centrifuging the supernatant at 4000r/min for 10 minutes, and filtering by a 0.22 mu m microporous filter to obtain a sample liquid containing phage;
(2) Preparing a host bacterium:
inoculating single colony of streptomycete with good growth on a flat plate into 100mL of Gao's liquid culture medium, and culturing at 170r/min and 30 ℃ until logarithmic growth is early for later use;
(3) Proliferation isolation of phages
Adding 10mL of a sample solution containing phage into 30mL of a Gao's first liquid culture medium, adding 1mL of a host bacterium suspension in the early logarithmic phase growth phase, adding a certain amount of calcium chloride, fully and uniformly mixing to ensure that the final concentration of the calcium chloride in the system is 10mmol/L, performing shake culture at 30 ℃ and 170r/min for overnight, centrifuging the obtained culture solution at 4 ℃ and 10000r/min for 10min, collecting supernatant, and filtering with a 0.22 mu m microporous filter to obtain phage enrichment liquid;
sucking 100 mu L of host bacterial liquid in a sterile super clean bench, uniformly coating the host bacterial liquid on a Gao's solid culture medium flat plate, standing for 15min, then sucking 100 mu L of phage enrichment liquid diluted 10-1000 times by SM liquid on the flat plate coated with the host bacterial liquid, uniformly coating the phage enrichment liquid, standing for 15min, and then inversely culturing overnight to form plaque;
(4) Purification of phages
Single plaque with uniform size and transparent is picked up by using sterile toothpick, placed in 1mL SM solution, and after shaking culture at 30deg.C and 170r/min, the filtrate is filtered, and diluted by sterile SM solution for 10 3 -10 6 After doubling, continuing to plate, purifying according to the operation, and repeating for a plurality of times to obtain purified plaque;
the evenly transparent single plaque is selected and placed in SM liquid, and after shaking culture and filtration, purified phage solution is obtained;
(5) Preservation of phages
The phage solution is placed in a refrigerator at 4 ℃ and can be stored for a long time, and is recovered every half year. Mixing phage solution with 60% glycerol at a ratio of 1:2, placing into a freezing tube, storing at-80deg.C for long term, and regularly recovering.
Naming said phage as
The phage 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 phage can form transparent plaque with uniform shape and size on a double-layer agar culture medium plate, no halo is arranged around the phage, the edge is clear and regular, and the diameter is about 3 mm; the phage should belong to the family Long tail phage (FIG. 1).
Example 2
Streptomyces scab phage
Potency and optimal multiplicity of infection determination
1. The titer of S.potato scab phage was determined:
phage titer refers to the number of infectious phage in each milliliter of sample. The phage titers were calculated as follows:
phage titer (PFU/mL) =number of plaques x dilution fold/phage liquid volume.
100 mu L of host bacteria liquid is sucked, the plate is coated and then is stood until the plate is dry, and 10 is diluted by sterile SM liquid 3 -10 6 100. Mu.L of phage solution was spread on a plate filled with host bacteria, cultured upside down at 30℃and the number of plaques on the plate was observed, and the test was repeated 3 times, 3 replicates each time.
2. Determination of optimal multiplicity of infection of phage
The multiplicity of infection (Multiplicity of Infection, MOI) refers to the ratio of the number of phages added to the host bacteria at the time of initial infection. 100uL of host bacteria and 100uL of phage are respectively added into a Gao's liquid culture medium according to the infection complex number of 100, 10, 1, 0.1, 0.01, 0.001 and 0.0001, shake culture is carried out at 30 ℃ and 170rpm, and a filter with 0.22um is used for filtering, thus obtaining pure phage solution. The phage solution is subjected to gradient dilution of 10 < -1 > to 10 < -9 >, the titer of phage is measured by using a flat-plate coating method, and the infection complex with the highest titer is the optimal infection complex. The test was repeated 3 times, 3 replicates each time.
Specifically, the results are shown in Table 1.
TABLE 1 phage
Optimal multiplicity of infection of (a)
The results showed that phage
At MOI of 0.01, the potency can reach 1.47×10 7 。
Example 3
Using phage (named
) The host profile of phage was determined by the spot method:
the 34 strains of streptomyces capable of causing scab were streaked on a plate, and single colony with good morphology was picked up for bacterial liquid culture. And (3) taking 100 mu l of bacterial liquid cultured until the early logarithmic growth phase, coating the bacterial liquid on an agar plate, taking 10 mu l of phage liquid stock solution by a pipetting gun to sample on the plate, paying attention to the interval between each drop of phage liquid stock solution when the bacterial liquid is sampled, completely drying the solution to be dripped, and culturing the bacterial liquid in an incubator at 30 ℃ in an inverted manner. The next day the plates were observed for the presence of plaques on their lawn, which indicates that the phage can lyse the bacteria. The details are shown in table 2 below.
TABLE 2 phage
Host range
The results showed that the phage
Has cleavage activity on S.scabies, S.galianeus and S.bobili. Said phage->
The obtained 34 strains of Streptomyces potato scab have lysis effect on 14 strains.
Example 4
Influence of temperature and pH on phage stability:
the potency reaches 10 8 Is placed in an EP tube after sterilization, is placed in a water bath kettle with different temperatures, is taken out after 1 hour, is immediately placed in an ice bath for cooling, and respectively measures phage titers at 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃, 90 ℃ and 100 ℃ by using a flat plate coating method. The test was repeated 3 times, 3 replicates each time.
SM solution with pH of 1-12 is prepared, and is filled into a sterile EP tube, and water bath is carried out at 30 ℃ for 30min until the temperature is balanced. To the potency of 10 8 100uL of phage are added into 900uL of SM solution with different pH values each time, water is put in a water bath kettle at 30 ℃ for 1h, an EP tube is taken out, and the titer of phage sample solution at different pH values is measured by a flat-plate coating method. The test was repeated 3 times, 3 replicates were set.
The results show that: said phage
Has activity in the pH range of 2-10, and has stable activity (figure 2). Phage activity was stable at 30℃to 80℃and inactivated at more than 80℃as shown in FIG. 3.
Example 5
One-step growth curve determination of phage:
mixing phage and host bacteria according to optimal infection complex, shaking at 30deg.C and 170rpm for adsorption for 15min, centrifuging at 10000rpm for 10min, discarding supernatant, re-suspending with fresh liquid culture medium, and repeatedly washing for 2-3 times to remove non-adsorbed phage. Adding 15mL of Gao's liquid culture medium, mixing, and rapidly placing in a shake incubator at 30deg.C for 170 r/min. Samples were taken continuously every 10min at intervals from time 0, sterile filtered, and the phage titers in the supernatants were determined by plate coating. And drawing a one-step growth curve, wherein phage titer is taken as an ordinate, and sampling time is taken as an abscissa. The test was repeated 3 times, 3 replicates each time. The results are shown in FIG. 4, phage
The incubation period of (2) was 20min and the burst period was 60min.
Example 6
Streptomyces scab phage
Prevention and treatment of potato scab:
100 seed potatoes with diameters of about 2cm and scab are selected and randomly divided into 2 groups. Soaking the disease seed potato in a range of 1×10 7 The phage suspension was incubated at 25℃for 24 hours and the other group was treated similarly except that the phage suspension was autoclaved at 121℃for 20min before soaking. The soaked potato seeds are planted in polystyrene foam boxes at the same depth, the foam boxes can freely leak water, the soil in the boxes is sterilized by steam at 60 ℃ for 1h, 5 plants are planted in each foam box, and 10 plants are repeated in each group. The foam box is placed in a greenhouse with the temperature of 22+/-2 ℃ and is watered every day within two weeks after planting, and the watering times are reduced after two weeks. Progeny tubers were harvested after 60d, and scab lesions were counted for each potato progeny tuber, and severity was assessed as percent lesion surface area and lesion type. Lesion surface area (%) was rated as follows on a scale of 0-6: 0, no lesions; 1, less than or equal to 10 percent; 2, 11-20%;3, 21-30%;4, 31-40%;5, 41-50%;6,50%. The type of damage is rated on a scale of 0-4 as follows: 0, no damage; 1. superficial lesions; 2. lesions that are slightly raised or depressed; 3. a recessed lesion; 4. deep concave lesions. The weight, size and tuber number of each plant were examined to determine the difference between the two groups.
The results are shown in Table 3.
TABLE 3 Streptomyces scab phage
Prevention and treatment effect on sub-potato scab
As can be seen from table 3: the control group had an average scab lesion coverage of around 28.5%, in contrast to a significant reduction in lesion coverage (3.5%) of tuber offspring of phage-treated group (p < 0.05). In addition, the type of injury (scab type) and the number of lesions (scab number) per tuber were also significantly reduced by phage treatment (p < 0.05). There was no significant difference in tuber weight, size or number between phage treated and untreated tubers
The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (9)
1. Streptomyces scab phage
The application of the streptomyces scab phage in preventing and controlling streptomyces scab is characterized in that
The preservation number of the steel is CCTCC NO: M2021379.
2. The use according to claim 1, characterized in that the phage of streptomyces scab
The separation and purification of (a) comprises the following steps:
(1) Sample pretreatment:
scraping the epidermis of the potato with the disease spots, grinding, adding distilled water, shaking and culturing at 30 ℃ and 170rmp for 6-8 hours, taking supernatant, centrifuging the supernatant at 4000r/min for 10 minutes, and filtering to obtain a sample liquid containing phage;
(2) Preparing a host bacterium:
inoculating single bacterial colony of streptomycete with good growth on a flat plate into a Gao's liquid culture medium, and culturing at 170r/min and 30 ℃ until logarithmic growth is early for later use;
(3) Proliferation isolation of phages
Adding 10mL of a sample solution containing phage into 30mL of a Gao's first liquid culture medium, adding 1mL of a host bacterium suspension in the early logarithmic phase growth phase, adding a certain amount of calcium chloride, fully and uniformly mixing to ensure that the final concentration of the calcium chloride in the system is 10mmol/L, performing shake culture at 30 ℃ and 170r/min overnight, obtaining a culture solution, centrifuging at 4 ℃ and 10000r/min for 10min, collecting supernatant, and filtering to obtain a phage enrichment solution;
sucking 100 mu L of host bacterial liquid in a sterile super clean bench, uniformly coating the host bacterial liquid on a Gao's solid culture medium flat plate, standing for 15min, then sucking 100 mu L of phage enrichment liquid diluted 10-1000 times by SM liquid on the flat plate coated with the host bacterial liquid, uniformly coating the phage enrichment liquid, standing for 15min, and then inversely culturing overnight to form plaque;
(4) Purification of phages
Single plaque with uniform size and transparent is picked up by using sterile toothpick, placed in 1mL SM solution, and after shaking culture at 30deg.C and 170r/min, the filtrate is filtered, and diluted by 10 by sterile SM solution 3 -10 6 After doubling, continuing to plate, purifying according to the operation, and repeating for a plurality of times to obtain purified plaque;
and (3) picking uniformly transparent single plaque, placing the plaque in SM solution, and obtaining purified phage solution after shake culture and filtration.
3. The use according to claim 2, wherein in step (2) the streptomyces is any one of s.scabies, s.galilaeus, s.bobili.
4. The use according to claim 2, wherein in step (4) the phage solution is stored at 4 ℃ and resuscitated every half year.
5. The use according to claim 2, wherein in step (4), the phage solution is mixed with 60% glycerol in a ratio of 1:2, and then placed in a freezing tube for long-term storage at-80 ℃ and periodically resuscitated.
6. A method for controlling scab, characterized in that the method comprises applying a Streptomyces scab phage to a plant
In which S.scab phage +.>
The preservation number of the steel is CCTCC NO: M2021379.
7. The control method of claim 6, wherein the plant is potato.
8. The control method according to claim 6 or 7, characterized in that the control method is immersing seed potato of potato in Streptomyces scab phage
The soaking time in the suspension is 24 hours.
9. The control method according to claim 6, wherein the scab is potato scab.
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