CN108034639A - Ralstonia solanacearum bacteriophage microecological preparation and preparation method and application thereof - Google Patents
Ralstonia solanacearum bacteriophage microecological preparation and preparation method and application thereof Download PDFInfo
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- CN108034639A CN108034639A CN201810119605.0A CN201810119605A CN108034639A CN 108034639 A CN108034639 A CN 108034639A CN 201810119605 A CN201810119605 A CN 201810119605A CN 108034639 A CN108034639 A CN 108034639A
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- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
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- C12N2795/00—Bacteriophages
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- C12N2795/00—Bacteriophages
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- C12N2795/00051—Methods of production or purification of viral material
Abstract
The invention provides a ralstonia solanacearum bacteriophage microecological preparation as well as a preparation method and application thereof, belonging to the technical field of microorganisms. The preparation method comprises the steps of inoculating ralstonia solanacearum liquid and ralstonia solanacearum phage liquid into a fermentation culture solution for fermentation culture; and when the fermentation culture is carried out for 8-20 h, feeding ralstonia solanacearum liquid into the host fermentation liquid, continuing the fermentation culture, controlling the whole fermentation period to be 24-48 h, and then inactivating the ralstonia solanacearum host to obtain the ralstonia solanacearum phage microecological preparation. According to the invention, fermentation feeding time is controlled to carry out fermentation culture for 8-20 h, and the host liquid is fed, so that the titer of the pseudomonas solanacearum phage can be greatly improved on the premise of not prolonging the fermentation time.
Description
Technical field
The invention belongs to microbial technology field, and in particular to a kind of ralstonia solanacearum bacteriophage probiotics and its preparation
Methods and applications.
Background technology
At present, the main means for controlling and eliminating pathogenic bacteria are still various methods physically or chemically, including various antibiosis
The use of element.Method physically or chemically all there is it is obvious the drawbacks of.First, elimination of the method for physics to various pathogenic bacteria
Effect can be only applied to a certain link of association area, it is impossible to realize the application of all links in whole field.Secondly, chemical method
Though soda acid processing in can obtain certain prevention effect, and after harmful bacteria forms biomembrane, the effect of these methods is with regard to pole
It is undesirable.The research and development of microbial ecological preparation are subject to each side to pay close attention to, and the research of particularly bacteriophage is favored be subject to people.
Since bacteriophage is found by Frederick Twort (1915) and F é lix DH é relle (1917), Europe and the former Soviet Union etc.
Country, has just just started to treat bacterium infection with bacteriophage.But in the emergence of the antibiotic such as penicillin, streptomysin, with
And preprophage drug failure it is fast the shortcomings of these medicines be eliminated soon.Although antibiotic begins to use effect to show
Write, but long-time service, it can not only strengthen the resistance to the action of a drug of pathogen, but also suppress profitable strain, cannot finally efficiently control,
Eliminate pathogenic bacteria.Late 1990s, many researchers, which are advocated, re-applies bacteriophage to clinical treatment, phagocytosis
Autogenic therapy is gradually recovered.In addition, bacteriophage has a wide range of applications in itself, such as Bacteria Identification and parting, for diagnosing
With treatment disease, the experimental tool of molecular biology research is also served as.Therefore, bacteriophage is widely applied value promotion bacteriophage
Fermenting and producing have been to be concerned by more and more people.
At present, the fermentation method for producing of bacteriophage has very much, for example, number of patent application is 94114314.7, it is entitled " to bite
The application for a patent for invention of the preparation method of thalline ecological agent " proposes a kind of medium culture method：In 10L aspirator bottles, liquid is filled
Bacteriophage is cultivated using host vibrios 5~7 days, final phage titer is 10 for 5000ml5~108PFU/ml.Number of patent application
200810145709.5 patent of invention uses host vibrios freeze-dried powder, and the potency of bacteriophage is not also high as nutrient source, be 1 ×
107PFU/ml.The life of bacteriophage is carried out in the patent of invention of number of patent application 200810202809.7 using Aeromonas hydrophila
Production, fermentation period length is 72~120h, and phage titer is 1 × 108PFU/ml.The hair of bacteriophage disclosed in above-mentioned patent
Fermenting process there are phage titer it is low the problem of.
The content of the invention
In view of this, it is an object of the invention to provide a kind of ralstonia solanacearum bacteriophage probiotics and preparation method thereof
And application, the preparation method ferment to obtain the ralstonia solanacearum bacteriophage of high-titer.
In order to realize foregoing invention purpose, the present invention provides following technical scheme：
The present invention provides a kind of preparation method of ralstonia solanacearum bacteriophage probiotics, comprise the following steps：
1) bacterial wilt bacterium solution is accessed in fermentation medium and cultivated, obtain host's zymotic fluid；Ralstonia solanacearum is in fermented and cultured
Initial cell concentration in base is 108~1014CFU/ml；
2) it is 10 by concentration10~1014The ralstonia solanacearum phagocytosis body fluid of PFU/ml, which is seeded in host's zymotic fluid, to ferment
Culture；The inoculum concentration of the ralstonia solanacearum phagocytosis body fluid is 1%~10%；
3) when fermented and cultured carries out 8~20h in the step 2), stream plus green grass or young crops in host's zymotic fluid into the step 2)
Blight bacterium solution, continues fermented and cultured, it is 24~48h to control whole fermentation period, obtains zymotic fluid；
4) zymotic fluid obtained in the step 3) is subjected to inactivation ralstonia solanacearum, obtains ralstonia solanacearum bacteriophage Tiny ecosystem
Preparation.
Preferably, the stream of bacterial wilt bacterium solution plus volume are the 1% of the volume of initial bacteriophage zymotic fluid in the step 3)
~10%；The concentration for the bacterial wilt bacterium solution that stream adds is 1010~1014PFU/ml。
Preferably, the volume of stream plus bacterial wilt bacterium solution is bacteriophage fermentating liquid volume per hour in the step 3)
0.7%~10%.
Preferably, in the step 3) between the stream added-time of bacterial wilt bacterium solution it is 6~12h.
Preferably, fermentation medium includes following content component in the step 1)：5~15g/L of peptone, beef extract powder
1~10g/L of 1~10g/L and sodium chloride；The pH value of the fermentation medium is 6.5~8.0.
Preferably, the fermentation culture conditions in the step 2) and step 3) are independently as follows：Fermentation temperature is 25~32 DEG C,
The pH value of host's zymotic fluid maintains 6~9；Fermentation carries out under agitation, and speed of agitator is 50~200rpm.
Preferably, the method for inactivation is water-bath in the step 4)；The temperature of water-bath is 45~70 DEG C；The time of water-bath is
2~5h.
The present invention provides a kind of microorganism state agent of ralstonia solanacearum bacteriophage, the potency of ralstonia solanacearum bacteriophage is 1013
~1016PFU/ml。
Green grass or young crops is identified or detected to probiotics the present invention provides the ralstonia solanacearum bacteriophage in plant protection
Application in blight bacterium.
It is the present invention provides a kind of preparation method of ralstonia solanacearum bacteriophage probiotics, bacterial wilt bacterium solution and green grass or young crops is withered
Germ phagocytosis body fluid, which is seeded in fermentation culture, carries out fermented and cultured；When fermented and cultured carries out 8~20h, ferment to host
Stream plus bacterial wilt bacterium solution in liquid, continue fermented and cultured, and whole fermentation period control is gone out in 24~48h, obtained zymotic fluid
Ralstonia solanacearum host living, obtains ralstonia solanacearum bacteriophage probiotics.The present invention is avoided after disposably adding bacterial wilt bacterium solution
Make the reduction of fermentation later stage host's bacteria concentration, and then influence the fermenting and producing of bacteriophage, flowed when fermented and cultured is carried out to 8~20h
Add supplement host's liquid, can realize the potency on the premise of not extending fermentation time, greatly improving bacteriophage.By different fermentations
The verification experimental verification of volume, using preparation method provided by the invention, the potency of the ralstonia solanacearum bacteriophage of fermenting and producing is 1013~
1016PFU/ml, compared with the method for the bacteriophage of prior art production, 2~6 orders of magnitude are improved in potency.
Meanwhile the inactivation treatment of zymotic fluid has been carried out in preparation method provided by the invention, ralstonia solanacearum host is gone out
It is living, the content of host vibrios in bacteriophage probiotics can be not only eliminated, but also be avoided that the harm to environment.
Further, preparation method provided by the invention, specifically defines the bar of fermentation medium component and fermented and cultured
Part, prepares ralstonia solanacearum bacteriophage microecological microbial agent with interflow plus the operation of host's zymotic fluid, can effectively further improve together
The potency of ralstonia solanacearum bacteriophage.
Embodiment
The present invention provides a kind of preparation method of ralstonia solanacearum bacteriophage probiotics, comprise the following steps：
1) bacterial wilt bacterium solution is accessed in fermentation medium and cultivated, obtain host's zymotic fluid；Ralstonia solanacearum is in fermented and cultured
Initial cell concentration in base is 108~1014CFU/ml；
2) it is 10 by concentration10~1014The ralstonia solanacearum phagocytosis body fluid of PFU/ml, which is seeded in host's zymotic fluid, to ferment
Culture；The inoculum concentration of the ralstonia solanacearum phagocytosis body fluid is 1%~10%；
3) when fermented and cultured carries out 8~20h in the step 2), stream plus green grass or young crops in host's zymotic fluid into the step 2)
Blight bacterium solution, continues fermented and cultured, it is 24~48h to control whole fermentation period, obtains zymotic fluid；
4) zymotic fluid obtained in the step 3) is subjected to inactivation ralstonia solanacearum host, it is micro- obtains ralstonia solanacearum bacteriophage
Ecological agent.
Bacterial wilt bacterium solution is accessed in fermentation medium and cultivated by the present invention, obtains host's zymotic fluid；Ralstonia solanacearum is fermenting
Initial cell concentration in culture medium is 108~1014CFU/ml。
The present invention is not particularly limited the method for the access, using access way well-known to those skilled in the art
.
In the present invention, the fermentation medium preferably includes following content component：5~15g/L of peptone, beef extract powder
1~10g/L of 1~10g/L and sodium chloride；More preferably 3~8g/ of 8~13g/L of peptone, 3~8g/L of beef extract powder and sodium chloride
L；Most preferably peptone 10g/L, beef extract powder 5g/L and sodium chloride 5g/L.The pH value of the fermentation medium is preferably 6.5
~8.0, more preferably 6.8~7.5, are most preferably 7.2.The present invention does not have special limit to the preparation method of the fermentation medium
System, using the preparation method of culture medium well-known to those skilled in the art.
In the present invention, after in the bacterial wilt bacterium solution access fermentation medium, the ralstonia solanacearum is in fermentation medium
In initial cell concentration be preferably 108~1014CFU/ml, more preferably 1010~1014CFU/ml, is most preferably 1012CFU/
ml.The present invention is not particularly limited the source of the ralstonia solanacearum, using ralstonia solanacearum well-known to those skilled in the art
Bacterial strain.Host's ralstonia solanacearum is enlarged culture by conventional method, obtains bacterial wilt bacterium solution.
After obtaining host's zymotic fluid, concentration is 10 by the present invention10~1014The ralstonia solanacearum phagocytosis body fluid inoculation of PFU/ml
The fermented and cultured into host's zymotic fluid；The inoculum concentration of ralstonia solanacearum phagocytosis body fluid is 1%~10%.
In the present invention, the source of the ralstonia solanacearum bacteriophage is not particularly limited, using those skilled in the art institute
Well known ralstonia solanacearum bacteriophage source.In the embodiment of the present invention, the ralstonia solanacearum bacteriophage separates from soil
Arrive.The present invention is not particularly limited the separated method, using bacteriophage separation side well-known to those skilled in the art
Method.
The present invention is not particularly limited the method for the inoculation, using access way well-known to those skilled in the art
.In the present invention, the concentration of ralstonia solanacearum phagocytosis body fluid is preferably 1011~1013PFU/ml, more preferably 1012PFU/
ml.The inoculum concentration of ralstonia solanacearum phagocytosis body fluid is preferably 3%~7%, and more preferably 5%.
In the present invention, the condition that fermented and cultured is carried out to the bacteriophage is preferably as follows：Fermentation temperature is 25~32 DEG C,
More preferably 28~30 DEG C.The pH value of host's zymotic fluid maintains 7~9, more preferably 7.5~8.5, is most preferably 8.0；Fermentation exists
Carried out under stirring condition, speed of agitator is 50~200rpm, more preferably 100~150rpm.
The present invention is when the fermented and cultured carries out 8~20h, stream plus bacterial wilt bacterium solution into host's zymotic fluid, after supervention
Ferment culture, it is 24~48h to control whole fermentation period, obtains zymotic fluid.
In the present invention, the stream of the bacterial wilt bacterium solution plus volume are preferably the 1%~10% of host's fermentating liquid volume, more
Preferably 2%~8%, it is most preferably 5%.The concentration of the bacterial wilt bacterium solution is 1010~1014PFU/ml。
In the present invention, the volume of stream plus bacterial wilt bacterium solution is the 0.5%~5% of host's fermentating liquid volume per hour, more
Preferably 1%~4%, it is most preferably 3%.
In the present invention, it is preferably 6~12h between the stream added-time of the bacterial wilt bacterium solution, more preferably 8h.
In the present invention, the condition for continuing fermented and cultured is preferably as follows：Fermentation temperature is 25~32 DEG C, more preferably
30℃.The pH value of host's zymotic fluid maintains 7~9, more preferably 7.5~8.5, is most preferably 8.0；Fermentation under agitation into
OK, speed of agitator is 50~200rpm, more preferably 100~150rpm.
The fermentation period of bacteriophage fermented and cultured is preferably 28~42h, more preferably 30~38h, is most preferably 35h.
After obtained zymotic fluid, the zymotic fluid is carried out inactivation ralstonia solanacearum host by the present invention, is obtained ralstonia solanacearum and is bitten
Thalline probiotics.
In the present invention, the method for the inactivation is preferably water-bath；The temperature of water-bath is preferably 45~70 DEG C, more preferably
50℃.The time of water-bath is preferably 2~5h, more preferably 3~4h.
The present invention provides a kind of probiotics of ralstonia solanacearum bacteriophage, the potency of ralstonia solanacearum bacteriophage is 1013
~1016PFU/ml。
In the present invention, the titration method of the ralstonia solanacearum bacteriophage, is measured with double-layer agar technique；The bilayer
Flat band method：1mL bacteriophage zymotic fluids are taken, 8000rpm centrifugation 20min, take 100 μ L of supernatant liquid, add 900 μ L sterile salines
In shake up, carry out gradient dilution successively.Take 10-12~10-14The dilution 100 μ L and 100 μ L of dilution gradient are in logarithmic growth
The host strain of phase is added to 8mL and is cooled in 50 DEG C or so of semisolid culturemedium, mixes, and 30 DEG C are inverted culture 12h, to plaque
Counted.The number of plaque and the calculation formula of potency are as follows：
Plaque number × extension rate × 10 on thalline potency (PFU/m L)=tablet.
The probiotics or the ralstonia solanacearum of the ralstonia solanacearum bacteriophage prepared the present invention provides the method
The application in ralstonia solanacearum is identified or detected to the probiotics of bacteriophage in plant protection.
A kind of ralstonia solanacearum bacteriophage probiotics provided by the invention and ralstonia solanacearum are bitten with reference to embodiment
Thalline fermentation preparation and application are described in detail, but they cannot be interpreted as the limit to the scope of the present invention
It is fixed.
Embodiment 1
Separated 3 plants of ralstonia solanacearum bacteriophages expand cultural method, comprise the following steps that：First measure the optimal sense of bacteriophage
Dye plural number：Infection multiplicity (multiplicityofinfection, MOI) refers to bite when bacteriophage primary infection host strain
The ratio of thalline quantity and host strain quantity, also referred to as infects multiple.In host strain inoculation 100ul to 200ml NB nutrient solutions,
150r/min 12h are cultivated to logarithmic phase.Then it is 10 to take initial potency10The bacteriophage nutrient solution of PFU/mL, according to infection multiplicity
Respectively 100,10,1,0.1,0.01,0.001,0.0001,0.00001,0.000001 ratio, by bacteriophage RS011 and φ
PRS011 is added in fluid nutrient medium with equal volume, 37 DEG C of shaking tables, 120r/min culture 10h, 4000r/min centrifugation 15min,
Remove precipitation, 0.22 μm of membrane filtration of supernatant, filter 23 time, with the potency of double-layer agar technique measure bacteriophage, potency highest
Infection multiplicity be optimal multiplicity of infection.The double-layer agar technique：1mL bacteriophage zymotic fluids are taken, 8000rpm centrifugation 20min, take
100 μ L of supernatant liquid, add in 900 μ L sterile salines and shake up, carry out gradient dilution successively.Take 10-12~10-14Dilution gradient
Dilution 100 μ L and 100 μ L be in the host strain of exponential phase and be added to the semisolid culturemedium that 8mL is cooled to 50 DEG C or so
In, mix, 30 DEG C are inverted culture 12h, and plaque is counted.It is inoculated with according to optimal multiplicity of infection, it is each of the total volume
2%, 33 DEG C, 150rpm, 18h is cultivated, obtains 3 plants of ralstonia solanacearum phagocytosis body fluid.
Embodiment 2
Stream plus the method for bacteriophage host fermenting and producing bacteriophage, comprise the following steps：
1) fermented using 50L fermentation tanks, the volume that fermentation medium is contained in fermentation tank is 35L, and host is accessed
In fermentation medium, it is 10 to make clump count in initial medium8CFU/ml；The fermentative medium formula is as follows：Peptone
15g/L；Beef extract powder 10gL；Sodium chloride 10g/L；PH is 8.0；
2) the 10 of the 1% of first plant of fermentating liquid volume are added10The ralstonia solanacearum phagocytosis body fluid of PFU/ml into fermentation tank into
Row culture, fermentation temperature are 28 DEG C, rotating speed 200rpm；PH value is controlled 7.5.
3) when fermentation carries out 8h, stream plus bacteriophage host into fermentation tank, controlled concentration is 1014Between CFU/ml, stream
It is 12h, flow acceleration 2L/h between added-time, stream adds up the amount of fermentation volume 6%, when fermentation period is 36 small, waits zymotic fluid face
When color becomes limpid by muddiness, you can put tank；
4) by the zymotic fluid of gained, the water-bath 5h under the conditions of 45 DEG C, inactivates host, up to bacteriophage probiotics.
5) the titration method of bacteriophage probiotics uses double-layer agar technique, plaque is counted, specifically
For：1mL bacteriophage zymotic fluids are taken, 8000rpm centrifugation 20min, take 100 μ L of supernatant liquid, add in 900 μ L sterile salines and shake
It is even, gradient dilution is carried out successively.Take 10-11~10-13The dilution 100 μ L and 100 μ L of dilution gradient are in exponential phase
Host strain is added to 8mL and is cooled in 50 DEG C or so of semisolid culturemedium, mixes, and 30 DEG C are inverted culture 12h, and plaque is carried out
Count.
The number of plaque and the calculation formula of potency are as follows：
Plaque number × extension rate × 10 on thalline potency (PFU/m L)=tablet.
Embodiment 3
Stream plus the method for bacteriophage host fermenting and producing bacteriophage, comprise the following steps：
1) fermented using 1000L fermentation tanks, the volume that fermentation medium is contained in fermentation tank is 800L, and host is connect
Enter in fermentation medium, the clump count for keeping initial medium is 1014CFU/mL.The fermentative medium formula is as follows：Albumen
Peptone 5g/L；Beef extract powder 1g/L；Sodium chloride 1g/L；PH is 6.5.
2) PFU for adding other one plant of fermentating liquid volume 5% is 1014The phagocytosis body fluid of PFU/ml is carried out into fermentation tank
Culture, fermentation temperature are 30 DEG C, rotating speed 200rpm；PH is controlled 6.5.
3) interior when fermentation progress 15 is small, stream plus bacteriophage host, controlled concentration is 1012CFU/ml is small for 8 between the stream added-time
When, flow acceleration 35L/h, stream adds up the amount of fermentation volume 8%, when fermentation period is 24 small, waits zymotic fluid color to present dark
During yellow, you can put tank.
4) by the zymotic fluid of gained, the water-bath 2h under the conditions of 60 DEG C, inactivates host, up to bacteriophage probiotics.
5) the titration method of bacteriophage probiotics uses double-layer agar technique, plaque is counted, specifically
For：1mL bacteriophage zymotic fluids are taken, 8000rpm centrifugation 20min, take 100 μ L of supernatant liquid, add in 900 μ L sterile salines and shake
It is even, gradient dilution is carried out successively.Take 10-11~10-13The dilution 100 μ L and 100 μ L of dilution gradient are in exponential phase
Host strain is added to 8mL and is cooled in 50 DEG C or so of semisolid culturemedium, mixes, and 30 DEG C are inverted culture 12h, and plaque is carried out
Count.
The number of plaque and the calculation formula of potency are as follows：
Plaque number × extension rate × 10 on thalline potency (PFU/m L)=tablet.
Embodiment 4
Stream plus the method for bacteriophage host fermenting and producing bacteriophage, comprise the following steps：
1) fermented using 5000L fermentation tanks, the volume that fermentation medium is contained in fermentation tank is 4000L, by host
Access in fermentation medium, the clump count for keeping initial medium is 1012CFU/ml；The fermentative medium formula is as follows：Egg
White peptone 10g/L；Beef extract powder 5g/L；Sodium chloride 5g/L；PH is 7.2.
2) PFU for adding the 3rd plant of fermentating liquid volume 1~10% is 1012The phagocytosis body fluid of PFU/ml into fermentation tank into
Row culture, fermentation temperature are 32 DEG C, rotating speed 150rpm；Control is 7.2.
3) interior when fermentation progress 20 is small, stream plus bacteriophage host, controlled concentration is 1013Between CFU/ml, between the stream added-time
For 6 it is small when, flow acceleration 200L/h, stream add up fermentation volume 10% amount, fermentation period for 48 it is small when, wait zymotic fluid color
When becoming limpid by muddiness, you can put tank.
4) by the zymotic fluid of gained, the water-bath 3h under the conditions of 55 DEG C, inactivates host, up to bacteriophage probiotics.
5) the titration method of bacteriophage probiotics is measured using double-layer agar technique, is specially：Take 1mL bacteriophages
Zymotic fluid, 8000rpm centrifugation 20min, takes 100 μ L of supernatant liquid, adds in 900 μ L sterile salines and shake up, carry out ladder successively
Degree dilution.Take 10-12The host strain that 100 μ L of the dilution and 100 μ L of dilution gradient are in exponential phase is added to 8mL and is cooled to
In 50 DEG C or so of semisolid culturemedium, mix, 30 DEG C are inverted culture 12h, and plaque is counted.
The number of plaque and the calculation formula of potency are as follows：
Plaque number × extension rate × 10 on thalline potency (PFU/m L)=tablet.
Comparative example 1
Scheme according to embodiment 4 is fermented, and difference is as follows for fermentative medium formula：Tryptone 10g/L,
Yeast extract 5g/L.The potency for the bacteriophage probiotics that fermentation obtains is counted and calculated according to the method for embodiment 4.
Comparative example 2
Scheme according to embodiment 4 is fermented, be a difference in that will stream plus bacteriophage host's liquid in step 2) together
Add in fermentation tank, the input constancy of volume of bacteriophage host's liquid.
The bioactivity of the bacteriophage probiotics of embodiment 2~4 and comparative example 1~2 the results are shown in Table 1.
The potency list of the bacteriophage probiotics of 1 embodiment 2~4 of table and comparative example 5
| Embodiment | Potency (PFU/ml) |
| Embodiment 2 | 1015 |
| Embodiment 3 | 1014 |
| Embodiment 4 | 1016 |
| Comparative example 1 | 109 |
| Comparative example 2 | 5×108 |
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (9)
1. a kind of preparation method of ralstonia solanacearum bacteriophage probiotics, it is characterised in that comprise the following steps：
1) bacterial wilt bacterium solution is accessed in fermentation medium and cultivated, obtain host's zymotic fluid；Ralstonia solanacearum is in the fermentation medium
Initial cell concentration is 108~1014CFU/ml；
2) it is 10 by concentration10~1014The ralstonia solanacearum phagocytosis body fluid of PFU/ml is seeded to fermented and cultured in host's zymotic fluid；
The inoculum concentration of ralstonia solanacearum phagocytosis body fluid is 1%~10%；
3) when fermented and cultured is carried out to 8~20h in the step 2), stream plus green grass or young crops are withered in host's zymotic fluid into the step 2)
Germ liquid, continues fermented and cultured, it is 24~48h to control whole fermentation period, obtains zymotic fluid；
4) zymotic fluid obtained in the step 3) is subjected to inactivation ralstonia solanacearum host, obtains ralstonia solanacearum bacteriophage Tiny ecosystem
Preparation.
2. preparation method according to claim 1, it is characterised in that the stream of bacterial wilt bacterium solution adds volume in the step 3)
For the 1%~10% of host's fermentating liquid volume；The concentration of bacterial wilt bacterium solution is 1010~1014PFU/ml。
3. preparation method according to claim 1, it is characterised in that stream adds bacterial wilt bacterium solution per hour in the step 3)
Volume be host's fermentating liquid volume 0.7%~10%.
4. according to the preparation method described in claims 1 to 3 any one, it is characterised in that ralstonia solanacearum in the step 3)
It is 6~12h between the stream added-time of liquid.
5. preparation method according to claim 1, it is characterised in that fermentation medium includes containing below in the step 1)
Measure component：1~10g/L of 5~15g/L of peptone, 1~10g/L of beef extract powder and sodium chloride；The pH value of the fermentation medium is
6.5~8.0.
6. preparation method according to claim 1, it is characterised in that the fermented and cultured bar in the step 2) and step 3)
Part is independently as follows：Fermentation temperature is 25~32 DEG C, and the pH value of host's zymotic fluid maintains 6~9；Fermentation carries out under agitation,
Speed of agitator is 50~200rpm.
7. according to the preparation method described in any one of claims 1 to 3,5 and 6, it is characterised in that inactivation in the step 4)
Method be water-bath；The temperature of water-bath is 45~70 DEG C；The time of water-bath is 2~5h.
8. ralstonia solanacearum bacteriophage probiotics prepared by claim 1~7 any one the method, it is characterised in that
The potency of ralstonia solanacearum bacteriophage is 1013~1016PFU/ml。
9. the ralstonia solanacearum bacteriophage probiotics described in claim 8 is identified or detected in ralstonia solanacearum in plant protection
Application.
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