CN114807056B - Virulent phage PSA-Pe and application thereof - Google Patents
Virulent phage PSA-Pe and application thereof Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
A virulent phage PSA-Pe and application thereof, wherein the virulent phage PSA-Pe is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) in the year 7 and 14 of 2020, and the preservation number is: CGMCC No.18920. The virulent phage PSA-Pe provided by the invention has the remarkable advantages that: (1) Can specifically kill pathogenic bacteria, and has quick action and long duration; (2) does not cause the generation of drug resistance of pathogenic bacteria; (3) The normal physiological metabolism of the plants is not influenced, the normal flora on soil and the plants is not influenced, and the method is nontoxic and harmless, ecologically friendly and environment-friendly; (4) As a specific phage of the pseudomonas syringae actinidia pathotype, the specific phage has strong specificity, can be applied to the control of the actinidia canker caused by the pseudomonas syringae actinidia Pathotype (PSA), and can be used for the rapid detection of pathogenic bacteria in plants, foods and environments; (5) can replicate by itself, and the research and development production cycle is short.
Description
Technical Field
The invention relates to the field of microbiology, in particular to a virulent phage PSA-Pe and application thereof, which is a virulent phage taking pseudomonas syringae and kiwi fruit pathotype as a host.
Background
The kiwi fruit canker occurs in all kiwi fruit producing areas of the world. Although the sources of pathogenic bacteria vary from place to place, genomic sequence analysis has shown that the pathogenic bacteria are Pseudomonas syringae, gram-negative bacteria, and pathogenic bacterial disease conditions are closely related to their host plants. Therefore, research on the incidence rules of ulcer diseases in different planting areas and development of efficient antibacterial preparations, especially biological preparations, effectively control the occurrence of ulcer diseases have realistic urgency and necessity.
The control of plant bacterial diseases is a worldwide problem, and traditionally, chemical pesticides are used as the main materials, and the disease can not be effectively controlled together with rotation and planting of disease-resistant varieties. The chemical pesticide control uses antibiotics and agricultural streptomycin as main components, so that bacteria propagating in plant tissues are difficult to achieve, bacteria are easy to generate drug resistance (tolerance) after long-term or large-scale use, super bacteria are generated, and environmental pollution and food safety problems are caused by pesticide residues. Therefore, the search for a novel and effective biological control method, "phage therapy," has become a hot spot of recent research.
Phage application to bacterial diseases or control of diseases can be traced back to the beginning of the twentieth century, however phage therapy has been ignored for a considerable period of time, accompanied by the discovery and widespread use of highly potent antibiotics. Twit in the United kingdom found phages in staphylococci for the first time in 1915. Phages are viruses that infect microorganisms such as bacteria and actinomycetes, and are the most numerous microorganisms on the earth so far, 10 times the number of bacteria. Phage maintain the balance of micro-ecology in nature by capturing bacteria. Phages can be classified into virulent phages and temperate phages according to their relationship to bacterial hosts. The former is also called as toxic phage, is a natural "killer" of bacteria, can replicate and proliferate in specific host bacteria cells, generates a plurality of progeny phage, and lyses bacteria, and the progeny phage can infect new host bacteria again, so as to further kill the host bacteria. Because the toxic phage has natural characteristics of natural schizomycete and tracking sterilization, and has no toxicity to plants, the corresponding toxic phage 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 for antibiotics to control bacterial diseases. It is reported that 75% of bacterial infections in the united states are now resistant to one or more antibiotics, and the therapeutic efficacy of antibiotics is increasingly diminished, particularly in the presence of "superbacteria", due to the continued emergence of resistant species as a result of abuse of antibiotics, while the development rate of new antibiotics is much slower than the rate at which bacteria overcome resistance to antibiotics, allowing phage use to re-stage the history.
Compared with the currently commonly used bacterial disease prevention and control mode, the phage has the following advantages: 1) The specificity is strong, and the ecological safety is good only for target pathogenic bacteria, and the effect on other microorganisms in the environment is avoided; 2) High efficiency: under the proper conditions, the phage can generate 200 filial generation in one lysis period, and the phage will have 200 parts n The proliferation is carried out at the speed of (2), and only a small amount of phage is needed for prevention and control; 3) Has the limitation of self-replication: they initiate a self-replication pattern by infecting host cells whenever host bacteria are present in the environment, and degrade when no host bacteria are present; 4) Is nontoxic to eukaryotic cells; 5) The method is not affected by bacterial multi-drug resistance, and can effectively prevent and control diseases caused by drug-resistant bacteria; 6) Bacteria have difficulty developing resistance to phage: the bacterial resistance mutation rate to antibiotics was 10 -6 While the mutation rate of resistance to phage was 10 -7 Mutation rate of combination antibiotic and phage administration was 10 -13 And the phage will produce the appropriate variation to accommodate the variation of the host bacteria; 7) The development period is short, the product can be stored for several months at 4 ℃, and the potency is not obviously reduced; 8) Can move along with bacteria to reach the inside of plant tissues, and has the effect that other medicaments can not prevent and treat.
One phage can only infect one bacterium, while one bacterium can be infected by a different phage. Therefore, phages with different sterilization effects can be separated and tested and applied to target flora in a combined way; by utilizing the specificity and parting of the phage, the phage rapid detection of pathogenic bacteria in plants, foods and environments is realized. Phage have also been isolated and utilized in plant bacterial disease studies with little success. For example, it is effective in phage isolation and control of potato black shank, cotton black branch, bacterial blight of corn, bacterial perforation of peach, bacterial wilt of tobacco, pear and raspberry pirimia, etc.
Disclosure of Invention
The present invention has two main purposes: firstly, a virulent phage PSA-Pe is provided, and secondly, a specific application of the virulent phage PSA-Pe is provided so as to make up for the defects of the prior art.
A virulent phage PSA-Pe deposited in the chinese microbiological bacterial culture collection center at 14, 7, 2020 under accession number: CGMCC No.18920. Preservation address: no.1 and No. 3 of the north cinquefoil of the morning sun area of beijing city. Suggested class naming: pseudomonas phage phi6 (Pseudomonas phage phi 6);
the virulent phage PSA-Pe is a saccular virus family tail-free phage capable of specifically cracking the pathogenic type of pseudomonas syringae and kiwi fruits, is spherical, has a spherical head, has no tail structure, and has a diameter and a length of 84+/-5 nm; incubation period is 75 minutes, lysis period is 35 minutes, burst size is 223, and stabilization period is reached 90-100 minutes after phage infection; the survival rate of phage is highest at pH7-8,4-37 ℃, and the phage activity is stable under natural temperature, pH value and UV-B irradiation conditions.
The phage is a virulent phage which can adapt to complex orchard environments.
The phage can be used to lyse pseudomonas syringae kiwi pathogenicity pathogenic PSA.
The phage can be used for rapid detection and identification of the pathotype of pseudomonas syringae kiwi fruits in plants, foods and environments.
The preparation method of the virulent phage PSA-Pe comprises the following steps:
(1) Isolation of kiwi fruit canker phage
The isolated PSA bacterial liquid is used as a host to be transferred into 20mL LB culture liquid, and is cultured for 16 hours at 25 ℃ under shaking at 150 rpm/min. Mixing the cultured PSA culture solution with sewage or soil samples, adding 200uL of the mixture into 10mL of 34-36 ℃ semisolid culture medium, rapidly pouring the mixture onto the thin solid culture medium cooled in advance, culturing at 25 ℃, and separating phage by a double-layer plate method.
(2) Purification, potency determination and preservation: single plaques were picked for inoculation, diluted, double-layered plates were re-cultured, plaques were counted, and phage purification and titer determination were performed. The purified phage can be directly placed in a refrigerator with the temperature of 4 ℃ for short-time preservation, and if phage liquid needs to be preserved for a long time, the phage liquid can be preserved according to the following steps of 1: adding 30% sterilized glycerol at a volume ratio of 1, mixing, sealing, storing at-80deg.C, and regularly recovering phage.
The phage can be used in the preparation of medicaments of pseudomonas syringae kiwi fruit pathogenic bacteria, in particular in the preparation of medicaments for treating diseases caused by PSA.
In addition, the phage can keep activity under the conditions of larger pH range, temperature and natural ultraviolet, which indicates that the phage can be used for controlling ulcer diseases in the complex climate environment of the kiwi fruit garden.
The invention has the advantages and technical effects that:
the phage provided by the invention has strong specificity and stronger environment adaptability, can be applied to the control of plant bacterial diseases caused by pseudomonas syringae kiwi pathotype, and has obvious advantages:
(1) The effect is obvious: under the proper conditions, the phage can generate 200 filial generation in one lysis period, and the phage will have 200 parts n The proliferation is carried out at the speed of (2), only a small amount of phage is needed for prevention and control, and the phage can move along with bacteria to reach the inside of plant tissues, so that the effect that other medicaments cannot prevent and control is achieved;
(2) The specificity is strong: can specifically kill pathogenic bacteria, and has quick action and long duration;
(3) Bacteria have difficulty developing resistance to phage: the bacterial strain is not influenced by bacterial multi-drug resistance, can effectively prevent and control diseases caused by drug-resistant bacteria, and has a resistance mutation rate of 10 to antibiotics -6 While the mutation rate of resistance to phage was 10 -7 Mutation rate of combination antibiotic and phage administration was 10 -13 And the phage will produce the appropriate variation to accommodate the variation of the host bacteria;
(4) Green and environment-friendly: the plant bacterial strain is only aimed at target pathogenic bacteria, can not affect other normal microorganisms in soil, plants and other environments, has the limitation of self replication, namely, the host bacteria are infected to start a self replication mode when the host bacteria exist in the environment, and are degraded when no host bacteria exist, so that the plant bacterial strain has good ecological safety, is nontoxic to eukaryotic cells, and does not affect normal physiological metabolism of the plants;
(5) The cost is lower: the phage related by the invention is obtained from nature, has large quantity, wide source, easy separation and acquisition, short research and development production period, belongs to natural organisms, can be self-replicated, can be stored for several months at 4 ℃, and has no obvious reduction in potency, so that the phage can be used as a therapeutic and preventive biological agent with low cost;
(6) As a pseudomonas syringae macaque specialization, the method can be used for rapidly detecting pathogenic bacteria in plants, foods and environments.
Drawings
FIG. 1 shows plaques in double-layer plate culture when phages are isolated according to the present invention.
FIG. 2 is a transmission electron microscope image of phage in the present invention.
FIG. 3 shows the growth curve of phage in the present invention.
FIG. 4 shows the activity curves of phages according to the invention at different temperatures.
FIG. 5 shows the activity curves of phages according to the invention at different pH values.
FIG. 6 shows the activity curves of phages according to the invention under different UV conditions.
Detailed Description
The technical solutions of the present invention will be clearly and completely explained and illustrated below with reference to the drawings and examples, and it is apparent that the described examples are only some of the examples of the present invention, but not all of the examples. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
The embodiment is a preparation method of the virulent phage PSA-Pe, which comprises the following steps:
(1) Isolation, purification and potency determination of kiwi fruit canker phage
The isolated PSA bacterial liquid is used as a host to be transferred into 20mL LB culture liquid, and is cultured for 16 hours at 25 ℃ under shaking at 150 rpm/min. Mixing the shaken PSA culture solution with sewage or soil samples, adding 200uL of the mixture into 10mL of semisolid culture medium, and putting the semisolid culture medium into a water bath in advance, wherein the temperature of the semisolid culture medium is kept between 34 and 36 ℃. Rapidly pouring onto a thin solid medium cooled in advance, separating phage by double-layer plate method, culturing at 25deg.C for 16 hr, and observing plaque formation.
After 16h incubation, if clear plaques were observed on the double-layered plate, single plaques were picked up and inoculated into 200uL of log-phase host bacterial liquid, placed in a 25℃incubator, and after 12h incubation with shaking at 150rpm/min, centrifuged at 12,000 rpm for 3min to obtain a supernatant, which was a pure culture liquid obtained by separating one phage. The phage pure culture solution filtered by using a 0.22uM aperture disposable filter is subjected to 10-time gradient dilution by using LB culture solution, 100uL of each dilution and 200uL of Psa bacterial solution are mixed and added into 20mL of LB culture solution, and the titer is measured by adopting a double-layer plate method.
Preservation of phage: the double-layer flat plate can be directly placed in a refrigerator at 4 ℃ for preservation in a short time, and taken out for phage activation in a fixed time. When the phage is preserved for a long time, under the aseptic condition, the purified phage culture solution is added into a 2mL aseptic preservation tube and mixed with glycerol according to the volume ratio of 1:1, the mixture is fully and uniformly mixed, sealed, preserved at the temperature of minus 80 ℃ and periodically resuscitated.
Electron microscope morphological observation of phage
The phage particles were negatively stained with a solution containing concentrated phage on a copper mesh and added with 2% uranyl acetate in water at ph=4.0 for 20s. The purified phage was observed with a JEM 1200EX transmission electron microscope and the image was scanned with a device camera as shown in FIG. 2. The phage was seen to have a spherical structure with only a spherical head and no tail structure, and it was found according to the guidelines of the international committee for classification of viruses that the phage was a tailless phage, which was a typical form of members of the family saccviridae, with a diameter and length of 92±5nm.
Growth characterization experiments
Determination of optimal multiplicity of infection MOI
First, by measuring the OD of a host bacterium grown in log phase 600 Value, and gradient dilution culture to count bacterial plaque, establish bacterial liquid OD 600 Relationship of value to its corresponding concentration. And counting plaques by phage dilution and double-layer plate culture, determining the concentration of phage, and calculating phage titer. The optimal MOI for phage purification or sterilization assays was determined to be 1, corresponding to titers of 2.32.+ -. 0.24X 10 12 PFU/mL, the optimal infection complex number can obviously improve the offspring cracking capacity of phage, and the order of magnitude of titer is improved.
Determination of one-step growth curve
To study the growth kinetics of phages, 5mL of phage culture broth was added to an equivalent host PSA bacterial culture at a ratio of MOI of 1, and the mixture was subjected to shaking culture at 25 ℃ for 5min, centrifuged at 8,000 rpm for 30s, the supernatant was decanted, and the tube bottom pellet was washed 2 times with LB broth. Then the thalli particles are resuspended in 10mL of LB, 150rpm/min and cultured in an oscillating way at 25 ℃; samples of 300uL were taken every 15min, phage titers were determined on double-layer plates for 150min, three replicates were averaged, and PSA broth without phage was set and phages were incubated under equivalent conditions as controls. The time of infection of host bacteria is taken as an abscissa, the logarithmic value of phage titer is taken as an ordinate, and the obtained one-step growth curve is shown in figure 3, and it can be seen that the incubation period of the phage is 75min, the lysis period is 35min, and the burst size is 150. Stabilization period is reached 90-100min after infection of phage.
Rapid detection and identification of pathogenic bacteria
Through sampling plant tissues, food or environment, the sample is crushed or directly stirred with sterile water, and bacteria in a water sample are conventionally separated and cultured. After the single colony is purified and cultured, 200 mu L of phage PSA-Pe with MOI of 1 is taken and mixed with 5mL of separated and purified bacteria, and double-layer flat plate culture is carried out at 25 ℃. And observing the generation and counting of the plaques, judging whether the pathotype and the content of the pseudomonas syringae kiwi fruits exist in the sample to be tested according to the existence and the quantity of the plaques, realizing the rapid detection and identification of the pathotype and providing predictive and early warning for the occurrence of diseases.
Example 2
The embodiment is a stability experiment of the virulent phage PSA-Pe, comprising the following steps:
(1) Thermal stability
To evaluate phage stability at different temperatures, experiments were performed in sterile LB at pH 7.0. The phage to be tested is placed into a water bath kettle for treatment at the temperature of 4 ℃,25 ℃,37 ℃ and 50 ℃ respectively, samples are taken at the temperature of 0h,3h,6h,12h and 24h respectively, the treated samples are immediately placed into an ice bath for cooling, LB culture solution is used for gradient dilution, phage titer is measured through a double-layer plate method, the phage is placed into a 25 ℃ incubator for culture for 16h, and three repeated average values are taken at each time point.
By counting plaques on the plate, phage titers were obtained, as shown in FIG. 4, which were stored at 4℃and 25℃for 24 hours at a concentration decrease of less than 0.16lg PFU/mL, and when the temperature was raised to 37℃the maximum inactivation amount of the experimental phage was 0.16lg PFU/mL and the maximum inactivation amount of the experimental phage at 50℃was 1.06lg PFU/mL, which means that even if high temperature was occasionally occurred, the survival of phage was not greatly affected. Thus, the phage PSA-Pe is relatively stable at 4-37 ℃.
(2) pH stability
To evaluate the pH stability of phage, LB culture media 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 was placed in a constant temperature incubator at 25℃and 0.5mL of each phage culture media was added after temperature equilibration, and the phage titer was measured by a double-layer plate method after 2 hours of treatment to detect changes in the activity. Three replicates were averaged for each treatment.
As shown in FIG. 5, when the phage PSA-Pe concentrations were tested at different pH values, a slight decrease in phage concentration was observed under both slightly acidic and slightly alkaline conditions, but the survival rate was highest between pH ranges of 7 and 8, and was relatively stable.
(3) Ultraviolet stability
Centrifuging phage liquid, diluting to 109PFU/mL, subpackaging into a centrifuge tube, placing the centrifuge tube under a UV-B ultraviolet lamp with the specification of 20W,50cm and 700mw/m2 for direct treatment at 30cm, respectively collecting the treated phage culture liquid at 0min,5min,15min,25min and 50min, balancing at dark place for 30min, performing gradient dilution with LB culture liquid, measuring phage titer, culturing in a culture box at 25 ℃ for 16h, and performing three repeated averaging for each group.
The titer is obtained by counting the plate plaques, a graph of the change of the phage titer with time is drawn, and the graph is shown in fig. 6, and the longer the phage is under the irradiation of a UV-B lamp, the lower the titer is, the activity is obviously reduced within 5min, the inflection point appears at 15min, a certain resistance is obtained, but the concentration of the phage is reduced by only 0.61lg PFU/mL, and the phage is relatively stable under the irradiation of the UV-B.
In summary, the phage PSA-Pe is active continuously and very stable under natural temperature, pH and UV-B irradiation conditions.
Claims (8)
1. A virulent phage PSA-Pe, characterized in that the virulent phage PSA-Pe is deposited in the chinese microbiological bacterial culture collection center under the accession number of the general microbiological center of the chinese microbiological bacterial culture collection center at 14, 7 months 2020: CGMCC No.18920.
2. The virulent phage PSA-Pe of claim 1, wherein the phage is spherical, has a spherical head, no tail, 92±5nm diameter, a latency of 72 minutes, a lysis period of 30 minutes, an outbreak size of 223, and reaches stationary phase 90-100 minutes after infection of phage.
3. The virulent phage PSA-Pe of claim 1, wherein the phage is capable of maintaining relatively stable sterilization activity at a pH ranging from 7-8, a temperature between 4-37 ℃, and uv-B.
4. Use of the virulent phage PSA-Pe according to claim 1 for the rapid detection of pathogenic bacteria in plants, food and the environment, which are pseudomonas syringae kiwi pathotype 3 variety PSA.
5. The use of the virulent phage PSA-Pe according to claim 1 for controlling conditions caused by pathogenic bacteria of Pseudomonas syringae kiwifruit, the phage titer being 10 1 -10 8 pfu/ml。
6. Use of the virulent phage PSA-Pe according to claim 1 for the preparation of a biological agent for a condition caused by pathogenic bacteria of pseudomonas syringae kiwi.
7. The use according to claim 6, wherein the biological agent is mixed with other phages isolated on the basis of pseudomonas syringae actinomycetes for the preparation of phage cocktails.
8. The method for preparing the virulent phage PSA-Pe according to claim 1, comprising the steps of:
(1) Isolation of kiwi fruit canker phage
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 hours h hours;
(2) Mixing the cultured PSA culture solution with sewage or soil samples, adding 200uL of the mixture into 10mL of prepared semisolid culture medium, rapidly pouring the culture medium onto a thin solid culture medium cooled in advance at 35 ℃, culturing at 25 ℃, and separating phage by a double-layer plate method;
(3) Purification and potency determination: selecting single plaque for inoculation, culturing the double-layer plate after dilution, counting the plaque, and carrying out phage purification and titer determination;
(4) And (3) preserving: the purified phage can be directly placed in a refrigerator with the temperature of 4 ℃ for short-time preservation; if long-term preservation of phage solution is required, the following steps are 1: adding 30% sterilized glycerol at a volume ratio of 1, mixing, sealing, storing at-80deg.C, and regularly recovering 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 |
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| 猕猴桃溃疡病菌噬菌体的初步研究;雷庆;叶华智;余中树;;安徽农业科学(19);全文 * |
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