CN114807056A - Virulent phage PSA-Pe and application thereof - Google Patents
Virulent phage PSA-Pe and application thereof Download PDFInfo
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Abstract
The virulent phage PSA-Pe is preserved in China general microbiological culture Collection center (CGMCC) at 7, 14 and 2020 with the preservation number as follows: CGMCC No. 18920. The virulent phage PSA-Pe provided by the invention has the following remarkable advantages: (1) the pathogenic bacteria can be specifically killed, the action time is short, and the duration is long; (2) the drug resistance of pathogenic bacteria can not be caused; (3) the normal physiological metabolism of plants is not influenced, the normal flora on soil and the plants is not influenced, and the fertilizer is non-toxic, harmless, ecological-friendly and environment-friendly; (4) the specific phage serving as pseudomonas syringae kiwi pathotype has strong specificity, can be applied to prevention and treatment of kiwi canker caused by pseudomonas syringae kiwi Pathotype (PSA), and can be used for rapid detection of pathogenic bacteria in plants, food and environment; (5) can be self-copied, and has short research and development period.
Description
Technical Field
The invention relates to the field of microbiology, in particular to a virulent phage PSA-Pe and application thereof, and the virulent phage is hosted by pseudomonas syringae kiwi pathotype.
Background
Kiwifruit canker occurs in every kiwifruit producing area in the world. Although the sources of pathogenic bacteria vary from place to place, genome sequence analysis shows that the pathogenic bacteria are Pseudomonas syringae (Pseudomonas) in the Pseudomonas genus and are gram-negative bacteria, and the pathogenic condition of the pathogenic bacteria is closely related to host plants of the pathogenic bacteria. Therefore, research on the incidence rules of the ulcer diseases in different planting areas, development of efficient antibacterial preparations, particularly biological preparations, and effective control of the incidence of the ulcer diseases are of real urgency and necessity.
The prevention and control of plant bacterial diseases are a worldwide problem, and traditionally, chemical pesticides are mainly used, and disease-resistant varieties are planted in a crop rotation mode, but the diseases cannot be effectively controlled. The chemical pesticide prevention and control mainly comprises antibiotics and agricultural streptomycin, so that bacteria propagating in plant tissues are difficult to achieve, the bacteria are easy to generate drug resistance (drug resistance) after long-term or large-scale use, super bacteria are generated, and the problems of environmental pollution and food safety are caused by pesticide residues. Therefore, the search for a new and effective biological control method, namely "phage therapy", has become a hot spot in recent years.
The application of bacteriophages for the control of bacterial diseases or illnesses dates back to the beginning of the twentieth century, however with the discovery and widespread use of highly potent antibiotics, phage therapy has been ignored for a considerable time. In 1915 Twort in UK, phages were first found in staphylococci. The phage is a virus infecting microorganisms such as bacteria and actinomycetes, and is a microorganism which has been most abundant on earth so far, and the number of the phage is 10 times that of the bacteria. Bacteriophages maintain the balance of microecology in nature by capturing bacteria. Bacteriophages can be classified into virulent bacteriophages and temperate bacteriophages according to their relationship to bacterial hosts. The former is also called as a toxic phage, which is a natural 'killer' of bacteria, and can replicate and proliferate in specific host bacteria cells to generate a plurality of progeny phages, and lyse bacteria, and the progeny phages can infect new host bacteria again to further kill the host bacteria. Because the toxic bacteriophage has natural characteristics of natural schizomycete and tracing sterilization, and has no toxicity to plant, the corresponding toxic bacteriophage for screening bacterial diseases can be applied to biological control of crop bacterial diseases.
At the end of the twentieth century, bacterial resistance has made it increasingly difficult to control bacterial diseases with antibiotics. It has been reported that 75% of the bacterial infections in the united states are now resistant to one or more antibiotics, and that the therapeutic efficacy of antibiotics is decreasing due to the emergence of resistant species caused by abuse of antibiotics, in particular the emergence of "superbacteria", whereas the development of new antibiotics is much slower than the rate at which bacteria overcome the resistance developed by antibiotics, so that the use of bacteriophages is re-embarked on the history stage.
Compared with the commonly used bacterial disease prevention and control mode at present, the bacteriophage has the following advantages: 1) the specificity is strong, only aiming at the target pathogenic flora, the influence on other microorganisms in the environment can not be generated, and the ecological safety is better; 2) high efficiency: under appropriate conditions, 200 progeny can be generated from a single lytic cycle of phage, and the phage will be exposed to 200 progeny n The proliferation is carried out at a speed, and only a small amount of phage is needed for prevention and treatment; 3) has the limitation of self-replication: as long asThey initiate a self-replicating mode by infecting host cells in the presence of host bacteria in the environment, and degrade in the absence of host bacteria; 4) is nontoxic to eukaryotic cells; 5) the method is not influenced by multiple drug resistance of bacteria, and can effectively prevent and control diseases caused by drug-resistant bacteria; 6) bacteria are very difficult to develop resistance to bacteriophages: the antibiotic resistance mutation rate of bacteria is 10 -6 And a resistance mutation rate to phage of 10 -7 The mutation rate of the antibiotic and phage combination is 10 -13 And the phage can generate appropriate variation to adapt to the variation of the host bacteria; 7) the period of development is short, the product can be stored for several months at 4 ℃, and the titer is not obviously reduced; 8) can reach the inside of plant tissues along with the movement of bacteria, and has the effect that other medicaments cannot prevent and treat.
One phage can only infect one bacterium, while one bacterium can be infected by a different phage. Therefore, the bacteriophage with different sterilization effects can be separated and tested, and can be jointly applied to the target flora; the specificity and the typing of the phage are utilized to realize the fast detection of the phage of pathogenic bacteria in plants, food and environment. There are also no very successful examples of the isolation and use of bacteriophages in the study of bacterial diseases in plants. For example, good effects are obtained in the separation and control of bacteriophages such as potato black shank, cotton black branch, corn bacterial blight, peach bacterial perforation, tobacco bacterial wilt, pear fire blight of apples, pears and raspberries.
Disclosure of Invention
The present invention has two main objects: the method is characterized in that one type of the virulent phage PSA-Pe is provided, and the other type of the virulent phage PSA-Pe is specifically applied to make up for the defects of the prior art.
The virulent phage PSA-Pe is preserved in China general microbiological culture Collection center (CGMCC) at 7-14.2020 with the preservation number: CGMCC No. 18920. And (4) storage address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North. Suggested classification nomenclature: pseudomonas phage phi6(Pseudomonas phage phi 6);
the virulent phage PSA-Pe is a capsoviridae tailless phage capable of specifically cracking pseudomonas syringae kiwi pathotype, is spherical, has a spherical head and a tailless structure, and has the diameter and the length of 84 +/-5 nm; the incubation period is 75 minutes, the lysis period is 35 minutes, the explosive amount is 223 minutes, and the stable period is reached 90-100 minutes after the bacteriophage is infected; the survival rate of the phage was highest at pH7-8, 4-37 ℃, and the activity of the phage continued to be very stable under natural temperature, pH and UV-B irradiation conditions.
The phage is a virulent phage capable of adapting to complex orchard environments.
The phage can be used for cracking pseudomonas syringae and kiwi pathogenic pathogen PSA.
The phage can be used for rapid detection and identification of pseudomonas syringae kiwi pathotype in plants, foods and environments.
The preparation method of the virulent phage PSA-Pe comprises the following steps:
(1) isolation of bacterial phage of kiwifruit canker
Transferring the separated PSA bacterial liquid as a host into 20mL LB culture solution, and carrying out shaking culture at 25 ℃ and 150rpm/min for 16 h. Mixing the cultured PSA culture solution with sewage or soil sample, adding 200uL mixture into prepared 10mL34-36 deg.C semisolid culture medium, quickly pouring onto the cooled thin layer solid culture medium, culturing at 25 deg.C, and separating phage by double-layer plate method.
(2) Purification, potency determination and preservation: selecting a single plaque for inoculation, diluting, culturing a double-layer plate, counting plaques, and purifying and measuring the titer of the phage. The purified phage can be directly stored in a refrigerator at 4 ℃ for a short time by inverting the double-layer plate, and if phage liquid needs to be stored for a long time, the ratio of the phage liquid to the purified phage liquid can be 1: adding 30% of sterilized glycerol in a volume ratio of 1%, mixing well, sealing, storing at-80 ℃, and periodically recovering the phage.
The phage can be used for preparing medicaments of pseudomonas syringae and kiwi pathotype bacteria, particularly medicaments for treating diseases caused by PSA.
In addition, the phage can keep activity under a larger pH range, temperature and natural ultraviolet conditions, which indicates that the phage can be used for controlling ulcer diseases in complex climatic environments of kiwi fruit orchards.
The invention has the advantages and technical effects that:
the phage provided by the invention has strong specificity and strong environment adaptability, can be applied to the prevention and treatment of plant bacterial diseases caused by pseudomonas syringae and kiwi pathotype, and has obvious advantages:
(1) the effect is remarkable: under appropriate conditions, 200 progeny can be generated from a single lytic cycle of phage, and the phage will be exposed to 200 progeny n The proliferation speed is high, only a small amount of phage is needed for prevention and treatment, and the phage can reach the interior of plant tissues along with the movement of bacteria, so that the effect that other medicaments cannot prevent and treat is achieved;
(2) the specificity is strong: can specifically kill pathogenic bacteria, and has quick action and long duration;
(3) bacteria are very difficult to develop resistance to bacteriophages: is not affected by multiple drug resistance of bacteria, can effectively prevent and control diseases caused by drug-resistant bacteria, and has a mutation rate of 10 -6 And a resistance mutation rate to phage of 10 -7 The mutation rate of the antibiotic and phage combination is 10 -13 And the phage can generate appropriate variation to adapt to the variation of the host bacteria;
(4) green and environment-friendly: aiming at the target pathogenic flora only, the method does not influence other normal microorganisms in the environments such as soil, plants and the like, and has self-replication limitation, namely the host bacteria can start a self-replication mode by infecting host cells as long as the host bacteria exist in the environment, and the host bacteria can degrade without existence of the host bacteria, so that the method has better ecological safety, is nontoxic to eukaryotic cells and does not influence the normal physiological metabolism of the plants;
(5) the cost is low: the phage related to the invention is obtained from nature, has large quantity, wide source, easy separation and acquisition, short development and production period, belongs to natural organisms, can be self-copied, can be stored for months at 4 ℃, and has no obvious reduction of potency, so the cost can be reduced when the phage is used as a biological preparation for treatment and prevention;
(6) as a pseudomonas syringae macaque specialization type, the kit can be used for quickly detecting pathogenic bacteria in plants, food and the environment.
Drawings
FIG. 1 shows plaques in double-layer plating when phages were isolated in the present invention.
FIG. 2 is a transmission electron microscope image of the bacteriophage of the present invention.
FIG. 3 is a graph showing the growth of the bacteriophage of the present invention.
FIG. 4 is a graph showing the activity of the bacteriophage at different temperatures according to the present invention.
FIG. 5 is a graph showing the activity of the bacteriophage at different pH values according to the present invention.
FIG. 6 is a graph showing the activity of the bacteriophage under different UV conditions according to the present invention.
Detailed Description
The technical solutions of the present invention will be clearly and completely explained and illustrated in the following drawings and embodiments, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
This example is a method for preparing bacteriophage PSA-Pe according to the present invention, comprising the following steps:
(1) separation and purification of kiwi fruit ulcer germ phage and titer determination
Transferring the separated PSA bacterial liquid as a host into 20mL LB culture solution, and carrying out shaking culture at 25 ℃ and 150rpm/min for 16 h. Mixing the shaken PSA culture solution with sewage or soil sample, adding 200uL of the mixture into 10mL of semisolid culture medium, putting the semisolid culture medium in a water bath kettle in advance, and keeping the temperature at 34-36 ℃. Quickly pouring the mixture on a thin-layer solid culture medium which is cooled in advance, namely separating the phage by adopting a double-layer plate method, and observing whether the plaque is formed after culturing for 16 hours at 25 ℃.
After culturing for 16h, if clear plaques are observed on the double-layer plate, picking a single plaque, inoculating the single plaque into 200uL log-phase host bacterial liquid, placing the single plaque into an incubator at 25 ℃, carrying out shaking culture at 150rpm/min for 12h, and then centrifuging the single plaque at 12,000 rpm for 3min to obtain a supernatant, namely a pure culture solution obtained by separating one phage. The phage pure culture solution filtered by a disposable filter with the aperture of 0.22uM is diluted by LB culture solution in a gradient of 10 times, 100uL of the phage pure culture solution and 200uL of Psa bacterial solution are sucked in each dilution and mixed and added into 20mL of LB culture solution, and the titer is measured by adopting a double-layer plate method for counting.
And (3) preservation of the phage: the double-layer flat plate can be directly placed in a refrigerator for storage at 4 ℃ in an inverted mode for short-time storage, and is taken out for phage activation after fixed time. During long-term storage, under the aseptic condition, adding the purified phage culture solution into a 2mL aseptic storage tube, mixing the phage culture solution with glycerol according to the volume ratio of 1:1, fully and uniformly mixing, sealing, storing at-80 ℃, and periodically recovering the phage.
Electron microscopy morphological observation of bacteriophages
The solution containing the concentrated phage was placed on a copper mesh, and the phage particles were negatively stained for 20s by adding a 2% uranyl acetate aqueous solution having a pH of 4.0. The purified phage were observed with JEM 1200EX Transmission Electron microscope and the image was scanned with the instrument camera as shown in FIG. 2. It can be seen that the phage structure is spherical and has only spherical head and no tail structure, and according to the guidelines of the International Commission on viral Classification, the phage belongs to the tailless phage, which is typically a member of the family capsoviridae, and has a diameter and length of 92. + -.5 nm.
Growth characteristic experiment
Determination of the optimal multiplicity of infection MOI
First, by measuring the OD of host bacteria growing in logarithmic phase 600 Value, and gradient dilution culture counting bacterial plaque, establishing OD of bacterial liquid 600 Value versus its corresponding concentration. And counting plaques by diluting the phage and culturing a double-layer plate, determining the concentration of the phage, and calculating the titer of the phage. The optimal MOI for phage purification or bactericidal assay was determined to be 1, corresponding to a potency of 2.32. + -. 0.24X 10 12 PFU/mL, optimal infectionThe number can obviously improve the progeny cracking amount of the phage, and the magnitude of the titer is improved.
Determination of one-step growth Curve
To study the growth kinetics of the phage, 5mL of phage culture broth was added to an equal amount of host PSA bacterial culture at an MOI of 1, incubated at 25 ℃ for 5min with shaking, the mixture was centrifuged at 8,000 rpm for 30s, the supernatant was decanted, and the tube bottom pellet was washed 2 times with LB medium. Then suspending the thallus particles in 10mL LB, 150rpm/min, and carrying out shaking culture at 25 ℃; sampling 300uL every 15min, measuring the titer of the phage by a double-layer plate for 150min in total, repeating for three times, taking an average value, and setting a PSA culture solution without the phage and a phage to be cultured under the same condition as a control. The time for infecting host bacteria is used as an abscissa, and the logarithm value of the titer of the phage is used as an ordinate, so as to obtain a one-step growth curve, as shown in fig. 3, the latency period of the phage is 75min, the lysis period is 35min, and the outbreak amount is 150. Stationary phase is reached 90-100min after phage infection.
Rapid detection and identification of pathogenic bacteria
Through sampling plant tissues, food or environment, crushing the sample or directly stirring the sample with sterile water, and conventionally separating and culturing bacteria in the water sample. After the single colony is purified and cultured, 200 mu L of phage PSA-Pe with the stored MOI of 1 is mixed with 5mL of separated and purified bacteria, and the mixture is cultured by a double-layer plate at 25 ℃. And observing the generation and counting of the plaques, judging the existence and the content of the pathogenic type and the content of the kiwi fruit of the pseudomonas syringae in the sample to be tested according to the existence and the quantity of the plaques, realizing the rapid detection and identification of pathogenic bacteria, and providing prediction and early warning for the occurrence of diseases.
Example 2
This example is a stability test of the virulent phage PSA-Pe according to the present invention, comprising the following steps:
(1) thermal stability
To evaluate the stability of the phages at different temperatures, the experiments were carried out in sterile LB at pH 7.0. The phage to be detected is respectively placed in a water bath for processing at 4 ℃, 25 ℃, 37 ℃ and 50 ℃, and is respectively sampled for 0h, 3h, 6h, 12h and 24h, the processed sample is immediately placed in an ice bath for cooling, LB culture solution is used for gradient dilution, the titer of the phage is measured by a double-layer plate method, the phage is placed in an incubator at 25 ℃ for culturing for 16h, and three repeated averages are made at each time point.
Phage titer was obtained by counting plaques on the plate, as shown in FIG. 4, which phage were stored at 4 ℃ and 25 ℃ for 24 hours with a concentration decrease of less than 0.16lg PFU/mL, with a maximum inactivation of 0.16lg PFU/mL for the experimental phage when the temperature was raised to 37 ℃ and 1.06lg PFU/mL for the experimental phage at 50 ℃, meaning that there was no significant effect on phage survival even when occasionally high temperatures occurred. Therefore, the phage PSA-Pe is stable at 4-37 ℃.
(2) Stability of pH
In order to evaluate the pH stability of the phage, LB culture solutions with pH gradients of 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, and 11.0 were prepared, 4.5mL of each solution was placed in a 25 ℃ incubator, 0.5mL of each solution was added after temperature equilibration, and after 2h of treatment, the titer of the phage was determined by a double-layer plate method to detect changes in activity. Three replicates were done for each treatment and the average was taken.
As shown in FIG. 5, when the concentration of PSA-Pe phage was tested at various pH values, a slight decrease in phage concentration was observed under both mildly acidic and mildly alkaline conditions, but the survival rate was highest between pH7 and 8, and was relatively stable.
(3) UV stability
Centrifuging the phage liquid, diluting to 109PFU/mL, subpackaging centrifuge tubes, placing the centrifuge tubes in a position 30cm below a UV-B ultraviolet lamp with specifications of 20W, 50cm and 700mw/m2 for direct injection treatment, collecting the treated phage culture solution in 0min, 5min, 15min, 25min and 50min respectively, placing the phage culture solution in a dark place for 30min for balancing, measuring the phage titer after gradient dilution by using LB culture solution, placing the phage culture solution in an incubator at 25 ℃ for culture for 16h, and performing three repeated averaging on each group.
The titer is obtained by counting the plaque of the plate, a curve graph of the titer of the phage changing along with time is drawn, as shown in figure 6, the longer the time is under the irradiation of the UV-B lamp, the lower the titer is, the activity is obviously reduced within 5min, and the inflection point appears at 15min, so that a certain resistance is obtained, but the reduction of the concentration of the phage is only 0.61lg PFU/mL, and the stability is relatively stable under the irradiation of the UV-B lamp.
In conclusion, the phage PSA-Pe has continuous activity and is very stable under the conditions of natural temperature, pH value and UV-B irradiation.
Claims (10)
1. The virulent phage PSA-Pe is preserved in China general microbiological culture Collection center (CGMCC) at 7, 14 and 2020 with the preservation number: CGMCC No. 18920.
2. The virulent phage PSA-Pe of claim 1, wherein said phage is a virulent phage PSA-Pe that specifically cleaves the pathogenic form of pseudomonas syringae kiwifruit.
3. The virulent phage PSA-Pe according to claim 2, wherein said phage is spherical, having only spherical heads, no tails, a diameter of 92 ± 5nm, a latency of 72 minutes, a lytic phase of 30 minutes, a burst size of 223, and a stationary phase reached 90-100 minutes after infecting the phage.
4. The virulent phage PSA-Pe according to claim 2, wherein the phage retains relatively stable bactericidal activity at a pH in the range of 7-8, at a temperature of 4-37 ℃, and under uv-B.
5. The bacteriophage PSA-Pe according to claim 2, wherein the pathogenic bacterium is pseudomonas syringae kiwifruit pathotype type variant type 3 PSA.
6. The use of the virulent bacteriophage PSA-Pe according to claim 2 for the rapid detection of pathogenic bacteria in plants, food and the environment.
7. The intensity of claim 5Application of bacteriophage PSA-Pe in prevention and treatment of diseases caused by pseudomonas syringae and kiwi pathogenic bacteria, wherein the titer of the bacteriophage is 10 1 -10 8 pfu/ml。
8. The use of the virulent bacteriophage PSA-Pe according to claim 7 for the preparation of a biological agent for the pathological conditions caused by pathogenic bacteria of pseudomonas putida kiwifruit.
9. The biological agent according to claim 8, wherein the biological agent is not limited to its use alone, and can be mixed with other phages isolated based on Pseudomonas syringae Kiwi pathotype bacteria to make phage cocktail preparations.
10. The method for producing bacteriophage PSA-Pe according to claim 1, comprising the steps of:
(1) isolation of bacterial phage of kiwifruit canker
Transferring the separated PSA bacterial liquid as a host into 20mL LB culture liquid, and carrying out shaking culture at 25 ℃ and 150rpm/min for 16 h;
(2) mixing the cultured PSA culture solution with sewage or a soil sample, adding 200uL of the mixture into a prepared 10mL semisolid culture medium, quickly pouring the mixture onto a thin-layer solid culture medium cooled in advance at the temperature of 35 ℃, culturing at 25 ℃, and separating phage by a double-layer plate method;
(3) purification and potency determination: selecting a single plaque for inoculation, diluting, culturing a double-layer flat plate, counting plaques, and purifying and measuring the titer of the phage;
(4) and (3) storage: the purified phage can be directly placed in a refrigerator at 4 ℃ upside down for short-time storage; if long-term preservation of phage liquid is required, the ratio of 1: adding 30% of sterilized glycerol in a volume ratio of 1%, mixing well, sealing, storing at-80 ℃, and periodically recovering the phage.
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| CN112359024A (en) * | 2020-11-14 | 2021-02-12 | 菲吉乐科(南京)生物科技有限公司 | Pseudomonas syringae bacteriophage and composition, kit and application thereof |
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| CN112359024A (en) * | 2020-11-14 | 2021-02-12 | 菲吉乐科(南京)生物科技有限公司 | Pseudomonas syringae bacteriophage and composition, kit and application thereof |
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| Title |
|---|
| 雷庆;叶华智;余中树;: "猕猴桃溃疡病菌噬菌体的初步研究", 安徽农业科学, no. 19 * |
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