CN108676719A - A kind of store method of Ralstonia solanacearum bacteriophage - Google Patents
A kind of store method of Ralstonia solanacearum bacteriophage Download PDFInfo
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- CN108676719A CN108676719A CN201810465069.XA CN201810465069A CN108676719A CN 108676719 A CN108676719 A CN 108676719A CN 201810465069 A CN201810465069 A CN 201810465069A CN 108676719 A CN108676719 A CN 108676719A
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- ralstonia solanacearum
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Abstract
The invention discloses a kind of store method of Ralstonia solanacearum bacteriophage, nontoxic Ralstonia solanacearum RSsw326 2 disclosed by the invention is deposited in China typical culture collection center, and deposit number is CCTCC NO:M 2018197.Nontoxic ralstonia solanacearum in the present invention is the ralstonia solanacearum detached from natural occurrence tobacco plant, the nontoxic Ralstonia solanacearum obtained by squamous subculture, the Ralstonia solanacearum mixing using bacteriophage stoste and equivalent used in this motion, and final concentration of 40% glycerine is added after 75 DEG C preserve 12 months, potency decline it is few, it is long to the holding time of Ralstonia solanacearum bacteriophage and higher activity can be kept.
Description
Technical field
The present invention relates to biotechnologies and microbial product field, are specifically related to a kind of preservation of Ralstonia solanacearum bacteriophage
Method.
Background technology
Bacteriophage（phage）It is the virus for invading bacterium, is also to confer to the inhereditary material of host strain biological character.Phagocytosis
Body can kill the phenomenon that bacterium 1915 by Frderic Te Wote (Frederick W.Twort) find, thus profit
The research for controlling bacterium with bacteriophage has last 100 years.Bacteriophage must have stringent host specificity in viable bacteria endoparasitism,
Molecular structure and complementarity depending on phage adsorption organ and recipient bacterium surface receptor.Bacteriophage is the most universal in virus
The most wide group with distribution.Usually in the place that some are full of bacterial community, such as：Soil, animal enteron aisle in, can find
Bacteriophage.With the development of molecular biology technology, bacteriophage is widely used for the multiple fields such as medical diagnosis on disease, water process.
The characteristic that gene can be inserted into due to bacteriophage in host DNA so that it also becomes important molecule and genetics research
Tool.Using bacteriophage, the experiment of many exquisitenesses can be designed.
However in above process, it is required for preserving bacteriophage strain, existing plant pathogenetic bacteria is especially green
The store method potency of withered bacterium bacteriophage declines mostly and the holding time is shorter, and effect is undesirable, seriously constrains and answers bacteriophage
Development.
Invention content
The purpose of the present invention is to provide a kind of store methods of Ralstonia solanacearum bacteriophage.
To achieve the above object, the present invention adopts the following technical scheme that：
The store method of Ralstonia solanacearum bacteriophage, by filtered bacteriophage stoste and the Ralstonia solanacearum suspension with volume ratio 1:
1 ratio mixing, is added final concentration of 40% glycerine, in -75 DEG C of Cord bloods.
A concentration of the 10 of the Ralstonia solanacearum suspension8 CFU /mL。
The Isolates of Pseudomonas Solanacearum Smith is that the Ralstonia solanacearum is eggplant Raul Salmonella（Ralstonia solanacearium）
RSsw326-2, is preserved in China typical culture collection center on April 12nd, 2018, and preserving number is CCTCC NO:M
2018197.Address is Wuhan University.
The advantage of the invention is that：
Final concentration of 40% glycerine is added in the Ralstonia solanacearum mixing of filtered Ralstonia solanacearum bacteriophage stoste and equivalent by the present invention,
In -75 DEG C of Cord bloods, bacteriophage still keeps higher activity.Store method operation rationally, conveniently, does not need special stabilization
Agent, and energy long-term preservation, the bacteriophage for being suitable for common laboratory preserve.
Description of the drawings
Fig. 1 bacterial strain RSsw326-2 bacterium colony figures.
Fig. 2 plaque figures.
Fig. 3 Ralstonia solanacearum specific primer 759F/760R PCR amplification figures（M：Marker 2000;1:Avirulent strains
RSsw326-2;2:Wild Ralstonia solanacearum）.
Specific implementation mode
Embodiment 1
1, the acquisition of ralstonia solanacearum：It from natually morbid tobacco plant, is detached using dilution spread partition method, picking single bacterium
Drop into 3-5 purifying of row, the wild Ralstonia solanacearum of acquisition.
2, the screening of avirulent strains：The wild ralstonia solanacearum detached from natural occurrence tobacco plant, by 20 more than generation
Continuous culture, the bacterium colony of picking poor fluidity carry out Pathogenicity, and the measurement result bacterial strain is lost to the pathogenic of tobacco,
The as dissociant of toxicity Ralstonia solanacearum, is named as RSsw326-2.
3, Pathogenicity：The single bacterium that picking squamous subculture obtains dissociant falls within shaken cultivation in NB fluid nutrient mediums
（28 DEG C, 180rpm）It is about 10 to concentration8CFU/mL is inoculated in the tobacco seedling of 5 true leaves using pouring root, after inoculation processing
30d, the tobacco being inoculated with do not show symptom as clear water compares inoculation, illustrate that obtained dissociant is lost to cigarette
Careless is pathogenic, as avirulent strains eggplant Raul Salmonella（Ralstonia solanacearium）RSsw326-2.
4, Ralstonia solanacearum specific primer PCR amplification：The genomic DNA for extracting avirulent strains, with Ralstonia solanacearum specific primer
759F/760R（GTCGCCGTCAACTCACTTTCC/GTCGCCGTCAGCAATGCGGAATCG）Carry out PCR amplification, amplification condition
For：95 DEG C of pre-degeneration, 5min；95 DEG C of denaturation, 30s；59 DEG C of annealing, 30s；Extend 72 DEG C, 1min；Extend 72 DEG C afterwards, 10min.
As a result it can get the specific band that a size is 280bp.See Fig. 3.
5, the utilization power of RSsw326-2 pairs of 3 kinds of disaccharide and 3 kinds of hexanols of avirulent strains：In basal medium（NH4H2PO4
1g、KCl 0.2 g、MgSO4·7H20.2 g of O, 1.0 g of peptone, phenol red 80 mg, 3 g of agar, pH7.4 is adjusted, added
Ionized water is settled to 1L）, it is separately added into the lactose of filtration sterilization, maltose, cellobiose, mannitol, sorbierite and sweet and pure,
Make its final concentration of 1%, dispense test tube, every pipe 3mL, by 1% ratio inoculation avirulent strains suspension（Concentration is about 108 CFU /
mL）, it is placed in 28 DEG C of constant incubators and cultivates.The result shows that the bacterial strain can utilize lactose, maltose, cellobiose, mannitol,
Sorbierite and sweet and pure.It is shown in Table 1.
Utilization powers of the 1 avirulent strains RSsw326-2 of table to trisaccharide triol
RSsw326-2 its bacterium colony on TTC culture mediums is round（See Fig. 1）, central kermesinus, edge milky, poor fluidity, lead to
The utilization power and Ralstonia solanacearum specific primer PCR amplification result for crossing 3 kinds of disaccharide of bacterial strain pair and 3 kinds of hexanols are identified as eggplant Raul
Salmonella（Ralstonia solanacearium）.
2 avirulent strains host range of embodiment measures：
Use topical application method avirulent strains host range.Concrete operations are：
Take 1mL logarithmic phase eggplant Raul Salmonellas（Ralstonia solanacearium）CPG semisolid culturemediums are added in culture
In, it is poured into after mixing on CPG solid agar tablets well prepared in advance, takes 0.5 μ L prophage drops to be added to after to be solidified flat
Plate surface, drying, 28 DEG C of inversion overnight incubations, 2d observe the formational situation of plaque.The result shows that the bacterial strain can be by green withered
Bacterium bacteriophage is parasitic（Fig. 2）.
Embodiment 3
（1）Bacterial strain RSsw326-2 is inoculated on CPG tablets, is inverted, 28 DEG C of culture 24-48h make it grow single bacterium colony.
（2）Picking bacterial strain RSsw326-2 single bacteriums are fallen in the conical flask equipped with 100mL NB culture mediums, 28 DEG C,
180rpm shaken cultivations to concentration is about 108cfu/ml。
（3）1mL bacteriophage stostes are added, mixing stands 15min, and 28 DEG C, 130rpm shaken overnight cultures keep bacterium complete
It totally cleaves solution.
（4）Liquid 12000rpm centrifugation 5min will be trained altogether, with 0.45 μm of membrane filtration supernatant to get to bacteriophage stoste.
（5）Cultured bacteriophage stoste is taken, its potency is measured and is recorded spare.
（6）The bacteriophage stoste of potency will have been surveyed（2.29*1010PFU /mL）Do following processing：1. stoste room temperature is protected
It deposits；2. 4 DEG C of preservations of stoste；3. -20 DEG C of preservations of stoste；4. -75 DEG C of preservations of stoste；5. the Ralstonia solanacearum of bacteriophage stoste and equivalent is mixed
It is even, and final concentration of 40% glycerine is added in -75 DEG C of preservations；
（7）3 months after preservation, measure its potency within 6 months, 9 months and 12 months.
Experimental result is shown in Table 2, time-to-live and survival condition difference, bacteriophage stoste of the different store methods to bacteriophage
It is preserved 3 months under room temperature, can't detect bacteriophage；Bacteriophage stoste preserves 12 months at 4 DEG C, and potency declines 5 orders of magnitude；
Bacteriophage stoste preserves 12 months at -20 DEG C and 75 DEG C, and potency declines 1 order of magnitude；And the 10 of bacteriophage stoste and equivalent8
CFU/mL bacterial strain RSsw326-2 mixings, and final concentration of 40% glycerine is added and is preserved 12 months in -75 DEG C, potency is in same number
Magnitude declines less, thus this kind of store method is long to the holding time of bacteriophage and can keep higher activity.
The titration of the different preserving type Ralstonia solanacearum bacteriophages of table 2
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with repair
Decorations should all belong to the covering scope of the present invention.
SEQUENCE LISTING
<110>University Of Agriculture and Forestry In Fujian
<120>A kind of store method of Ralstonia solanacearum bacteriophage
<130> 2
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213>Artificial sequence
<400> 1
gtcgccgtca actcactttc c 21
<210> 2
<211> 24
<212> DNA
<213>Artificial sequence
<400> 2
gtcgccgtca gcaatgcgga atcg 24
Claims (3)
1. a kind of store method of Ralstonia solanacearum bacteriophage, it is characterised in that：Filtered bacteriophage stoste and Ralstonia solanacearum are suspended
Liquid is with volume ratio 1:1 ratio mixing, is added final concentration of 40% glycerine, in -75 DEG C of Cord bloods.
2. a kind of store method of Ralstonia solanacearum bacteriophage according to claim 1, it is characterised in that：The Ralstonia solanacearum suspends
A concentration of the 10 of liquid8 CFU /mL。
3. a kind of store method of Ralstonia solanacearum bacteriophage according to claim 1, it is characterised in that：The Ralstonia solanacearum is
Eggplant Raul Salmonella（Ralstonia solanacearium）RSsw326-2 is preserved in Chinese Typical Representative on April 12nd, 2018
Culture collection, preserving number are CCTCC NO:M 2018197.
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| CN201810465069.XA CN108676719B (en) | 2018-05-16 | 2018-05-16 | Method for preserving ralstonia solanacearum bacteriophage |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113150994A (en) * | 2021-04-15 | 2021-07-23 | 上海理工大学 | Low-temperature preservation method for virulent phage |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0172914B1 (en) * | 1984-02-10 | 1990-05-16 | Japan Tobacco Inc. | Pseudomonas solanacearum m4s strain, composition containing the same for controlling soil-borne diseases of solanaceous plant, and method for controlling said soil-borne diseases with the same |
| CN104560891A (en) * | 2015-01-08 | 2015-04-29 | 贵州省烟草科学研究院 | Screening method of pseudomonas solanacearum bacteriophage resource |
-
2018
- 2018-05-16 CN CN201810465069.XA patent/CN108676719B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0172914B1 (en) * | 1984-02-10 | 1990-05-16 | Japan Tobacco Inc. | Pseudomonas solanacearum m4s strain, composition containing the same for controlling soil-borne diseases of solanaceous plant, and method for controlling said soil-borne diseases with the same |
| CN104560891A (en) * | 2015-01-08 | 2015-04-29 | 贵州省烟草科学研究院 | Screening method of pseudomonas solanacearum bacteriophage resource |
Non-Patent Citations (3)
| Title |
|---|
| 王羽: "无致病力青枯病病菌对番茄青枯病的控病研究", 《中国优秀硕士学位论文全文数据库》 * |
| 苏靖芳等: "青枯雷尔氏菌噬菌体研究进展", 《土壤与作物》 * |
| 高苗等: "一株裂解性青枯雷尔氏菌噬菌体的分离及生物学特性分析", 《中国农业科学》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113150994A (en) * | 2021-04-15 | 2021-07-23 | 上海理工大学 | Low-temperature preservation method for virulent phage |
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