CN105936879B - Bacillus subtilis K13 and its cultural method and application - Google Patents
Bacillus subtilis K13 and its cultural method and application Download PDFInfo
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Abstract
The invention discloses a kind of bacillus subtilis (Bacillus subtilis) K13 for inhibiting anthracnose of rubber trees bacterium; China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved on May 27th, 2015; deposit number is CGMCC No.10917, belongs to technical field of plant protection.Also disclose cultural method and the application of the bacillus subtilis (Bacillus subtilis) K13, bacillus subtilis (Bacillus subtilis) the K13 bacterial strain is through obtained by plate face-off experiment screening, the growth of anthracnose of rubber trees bacterium can effectively be inhibited, to person poultry harmless, green safe, environment compatibility is good, is not easy to produce resistance, it is able to carry out business development, is had a extensive future.
Description
Technical field
The invention belongs to technical field of plant protection, and in particular to bacillus subtilis (Bacillus subtilis) K13
And its cultural method and application.
Background technique
The right rubber of d is important strategic materials, belongs to a kind of property resource in short supply.It is listed as with iron ore, petroleum, coal
The four big raw materials of industry.All play huge effect in traffic and national defense industry, at the same it again with national economy situation, economy
Period and financial and monetary policy and international trade policy are closely connected；Therefore the right ruber plantation of d has very important
Status.The production process of the right rubber of d is influenced as agricultural product by the factors such as season and d gas, in the agricultural development of China hot-zone,
Compare the producing region of concentration, local farmers and Farm workers using the right rubber planting of d as the pillar industry increased income, greatly in the right rubber of d
There are about the plant developments that more than 300 ten thousand people are engaged in the right rubber of d.The right Rubber Industry of d all provides for the economic construction in China, national defense safety
Powerful support.For the economic development of each department, harmony stabilization has all made huge contribution.
Rubber anthracnose is a kind of important disease on rubber tree, and the rubber anthracnose state of an illness is serious year by year, up to now for
Only, which has generation in each Zhi Jiao state, the world.From nursery seedling, crop field treelet up at age open rubber tapping tree can be by anthrax
Disease causes harm.The germ infects tender leaf, petiole, tender tip and the fruit of rubber tree, causes tender leaf to fall off when serious, and tender tip returns withered and fruit
It is real to rot, or even form mummy suspension in the tree.
Rubber anthracnose finds that in recent years, domestic Rubber Industry situation occurs from Petch in 1906 for the first time in Sri Lanka
Significant change, the diversification of plantation, cultivation, management mode etc., and also the change of ecological environment, rubber important Defect inspection,
There are some new problems on monitoring and Control Technology.Such as, disease occur, catastrophic condition change, disease epidemic situation, occur and
Regularty of epidemic research lacks systematicness, and basic research work is weak；Disease monitoring, monitoring and warning operating technology underbraced；Newly
Occur and catastrophe risk disease lacks understanding and technological reserve；Chemical prevention and control method is single, there is ecology and drug resistance safety
Hidden danger；Disease-resistant rubber germ plasm resource situation is unclear, and the excavation and utilization of disease resistence gene resource are huge in disease control
Effect is not fully exerted.
In conclusion needing the feature for rubber anthracnose, a kind of control method for specially controlling rubber anthracnose is developed,
It fills up in place of the deficiencies in the prior art.
Summary of the invention
It is an object of the present invention to be directed to the prior art blank, provide it is a kind of inhibit anthracnose of rubber trees bacterium it is withered
Careless bacillus (Bacillus subtilis) K13, bacillus subtilis (Bacillus subtilis) K13 are a kind of
Separation screening obtains the biocontrol bacteria bacterial strain for having antagonism to anthracnose of rubber trees bacterium out of rubber tree, to rubber tree anthrax
The prevention and treatment of disease is of great significance.
Another object of the present invention is to provide the bacillus subtilis (Bacillus for inhibiting anthracnose of rubber trees bacterium
Subtilis) separation method of K13 and the application in terms of preventing and treating anthracnose disaster.
The present invention adopts the following technical scheme that achieve the goals above
Bacillus subtilis (Bacillus subtilis) K13, deposit number are as follows: CGMCC No.10917, in 2015
On May 27, in is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address are as follows: court, Beijing
The institute 3 of positive area's North Star West Road 1.
Further, the withered bacillus K13 is isolated from rubber leaf.
The cultural method of above-mentioned bacillus subtilis (Bacillus subtilis) K13, includes the following steps:
The bacillus subtilis is inoculated in shaken cultivation in LB liquid medium to obtain；
The formula of the LB liquid medium: contain in every 1000mL water: tryptone 10g, yeast extract 5g, NaCl
5g；PH is 7.4.
Further, include the following steps:
Step 1: the bacillus subtilis is accessed into potato dextrose agar and is sealed, constant temperature incubation obtains single bacterium
It falls；
Step 2: taking the single colonie to be forwarded in LB liquid medium, and sealing is placed in shake culture on shaking table, then
The LB liquid medium is transferred on LB solid medium, is smeared uniformly, constant temperature incubation, is obtained by infinite dilution method single
One bacterial strain；
Step 3: opposite culture is carried out to the single bacterial strain respectively: taking the bacteria cake of anthrax-bacilus RC178, is placed in LB plate
Among culture medium, it is inoculated with the single bacterial strain around the bacteria cake, seals, constant temperature incubation, according to the single bacterial strain to charcoal
The bacteriostasis of subcutaneous ulcer bacterium RC178, screening obtain bacillus subtilis (Bacillus subtilis) K13；
Step 4: the bacillus subtilis (Bacillus subtilis) K13 is taken to vibrate training in LB liquid medium
It supports, obtains bacillus subtilis (Bacillus subtilis) K13 strain.
Further, include the following steps: in the step 1: the constant temperature incubation condition are as follows: by the culture after inoculation
Base is inverted into constant incubator and cultivates, and temperature is 28 DEG C, incubation time 48h.
Further, include the following steps: in the step 2: the constant temperature incubation condition are as follows: by LB solid medium
Being inverted into the temperature cultivated in constant incubator is 28 DEG C, incubation time 48h, and being placed in the temperature of shake culture on shaking table is
28 DEG C, incubation time 12h.
Further, include the following steps: in the step 3: the LB plating medium after inoculation is inverted into constant temperature
The temperature cultivated in incubator is 28 DEG C, and incubation time is 4~7d；The diameter of the bacteria cake is 6mm；The opposite culture setting
3 repetitions are not inoculated with the single bacterial strain to be only inoculated with the anthrax-bacilus RC178 as control；
The screening technique are as follows: use two o'clock opposite culture；The Antagonistic Fungi of 28 DEG C of constant temperature incubation 7d of picking and pathogen
Homogeneity equivalent mycelia is inoculated on 2 points of distance PDA plate center 1.5cm, then is placed in 28 DEG C of insulating boxs and cultivates, day by day
Observe bacteriostasis；Above-mentioned opposite culture mode sets 3 repetitions, to be only inoculated with the plate of disease fungus as control.
The gene order of bacillus subtilis (Bacillus subtilis) K13 is as shown in SEQ ID NO 1.
The application of above-mentioned bacillus subtilis (Bacillus subtilis) K13, the bacillus subtilis
(Bacillus subtilis) K13 is used to prepare the biochemical reagents for having antagonism to anthracnose of rubber trees bacterium；It is used to prepare pair
Anthracnose of rubber trees bacterium mycelia has the biochemical reagents of inhibition, teratogenesis；It is used to prepare mitogenetic to anthracnose of rubber trees bacterium mycelia
Spore germination has the biochemical reagents of inhibiting effect.
The beneficial effects of the present invention are:
1, bacillus subtilis (Bacillus subtilis) K13 is endophyte of plant, is inhibited to anthracnose of rubber trees bacterium
Effect is strong, has antagonism to a variety of disease fungus, there is antagonistic bacterium to play in biocontrol of plant disease very heavy
The effect wanted, such as: type and quantity are numerous, largely exist in plant phyllospheric, leaf；It is a variety of to the mode of action of pathogen more
Sample, such as: there is antagonism to anthracnose of rubber trees bacterium, has inhibition, teratogenesis to anthracnose of rubber trees bacterium mycelia；To rubber
Tree anthrax bacteria mycelia conidia germination has inhibiting effect.Reproduction speed is fast, has to the prevention and treatment of anthracnose of rubber trees important
Meaning.
2, bacillus subtilis (Bacillus subtilis) K13 is strong to anthracnose of rubber trees bacterium inhibiting effect, is conducive to
The beneficial microorganism ecological balance is maintained, the antibacterial substance as caused by biocontrol bacteria is typically directly directed to corresponding pathogen
It works, high specificity helps to maintain so will not have an adverse effect to beneficial microbes other in Agro-ecological System
The beneficial microorganism ecological balance.
3, strain culturing condition is simple, easy to maintain, easy to industrialized production, with good development and application prospects.
Bacillus subtilis (Bacillus subtilis) K13:
Deposit number are as follows: CGMCC No.10917；
Preservation date: on May 27th, 2015；
Depositary institution's title: China Committee for Culture Collection of Microorganisms's common micro-organisms center；
Preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
Detailed description of the invention
Fig. 1 is plate face-off antagonism of bacillus subtilis (Bacillus subtilis) K13 to anthracnose of rubber trees bacterium
Action diagram；(a) it is the result 1 of bacillus subtilis (Bacillus subtilis) K13 and anthrax-bacilus RC178 face-off, (b) is
The result 2 of bacillus subtilis (Bacillus subtilis) K13 and anthrax-bacilus RC178 face-off (c) is bacillus subtilis
The result 3 of (Bacillus subtilis) K13 and anthrax-bacilus RC178 face-off (d) is control group --- the training of anthrax-bacilus RC178
Support result；
Fig. 2 is the growth curve of bacillus subtilis (Bacillus subtilis) K13 bacterial strain.
Specific embodiment
Below in conjunction with attached drawing detailed description of the present invention embodiment.It should be noted that described in following embodiments
The combination of technical characteristic or technical characteristic is not construed as isolated, they can be combined with each other to reaching more preferable
Technical effect.
Bacillus subtilis (Bacillus subtilis) K13, deposit number are as follows: CGMCC No.10917, in 2015
On May 27, in is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address are as follows: court, Beijing
The institute 3 of positive area's North Star West Road 1.The withered bacillus K13 is isolated from rubber leaf.
The application of above-mentioned bacillus subtilis (Bacillus subtilis) K13: the bacillus subtilis
(Bacillus subtilis) K13 is used to prepare the biochemical reagents for having antagonism to anthracnose of rubber trees bacterium；It is used to prepare pair
Anthracnose of rubber trees bacterium mycelia has the biochemical reagents of inhibition, teratogenesis；It is used to prepare mitogenetic to anthracnose of rubber trees bacterium mycelia
Spore germination has the biochemical reagents of inhibiting effect.
1 bacillus subtilis of embodiment (Bacillus subtilis) K13
1, it samples: winning the blade of anthrax scab from rubber tree, wash away the dust gravel on blade with clear water, dry in the air
It is dry, it is spare in the strong intersection clip minor illness leaf of disease with scissors；The size of clip minor illness leaf is 5mm × 5mm.
2, sample sterilizes: the minor illness leaf being put into beaker, 30s is impregnated with mercuric chloride, is during which constantly gently agitated for, then
Three times with aseptic water washing, it dries, the minor illness leaf after being sterilized.
3, bacillus subtilis (Bacillus subtilis) K13 is separately cultured: will be after disinfection with sterilized tweezers
Minor illness leaf is placed on potato dextrose agar solid (PDA) culture medium, and every ware uniformly puts 4 pieces, then culture dish is protected
Fresh film sealing, is inverted into constant incubator, and 28 DEG C of cultures are observed every 12h, the thallus grown is transferred after 48h
On to another potato dextrose agar solid medium, it is inverted into constant incubator, is carried out after 28 DEG C of culture 48h pure
Change, obtains bacillus subtilis (Bacillus subtilis) K13 after purification.
Using gradient dilution method, described bacillus subtilis (Bacillus subtilis) K13 after purification is applied respectively
Cloth is inverted into constant incubator in different LB plating mediums, and 28 DEG C of culture 48h take out above-mentioned LB plate respectively
Culture medium picks them separately on the slant medium that single colonie is seeded in different test tubes, is placed in incubator and saves, temperature 8
~10 DEG C.
4, the purifying of bacillus subtilis (Bacillus subtilis) K13
In aseptic operating platform, LB liquid medium is dispensed into conical flask, takes out the examination saved in the incubator
Pipe, is inoculated in the conical flask respectively with a small amount of bacillus subtilis of transfer needle picking (Bacillus subtilis) K13,
Sealing is placed in shake culture on shaking table, and temperature is 28 DEG C, after cultivating 12h, takes out the conical flask of shake culture on the shaking table,
200 microlitres are drawn on LB culture medium with pipettor, are smeared uniformly with paint daubs, are inverted into 28 DEG C of constant incubator cultures,
Incubation time is 48h, then obtains 29 plants of single bacterial strains by the processing of infinite dilution method；
5, the screening and culture of bacillus subtilis (Bacillus subtilis) K13
Following opposite culture is carried out respectively to 29 plants of single bacterial strains:
The bacteria cake for taking anthrax-bacilus RC178 is beaten with aseptic card punch, the diameter of bacteria cake is 6mm, is placed in LB plating medium
Between, the diameter of LB plate culture dish is 90mm, and 4 corners of the right-angled intersection around the bacteria cake are inoculated with the single bacterial strain,
3 repetitions are set, and sealing up culture dish with preservative film prevents from polluting.
Using dotted line facture and other face-off forms, opposite culture is carried out；To be only inoculated with the anthrax-bacilus RC178, no
Being inoculated with the single bacterial strain is control group.It is placed in constant incubator and cultivates, temperature is 28 DEG C, and incubation time is 4~7d.
According to the single bacterial strain to the bacteriostasis of anthrax-bacilus RC178, screening obtains bacillus subtilis (Bacillus
subtilis)K13；Screening technique are as follows: use two o'clock opposite culture；The Antagonistic Fungi of 28 DEG C of constant temperature incubation 7d of picking and pathogen
Homogeneity equivalent mycelia is inoculated on 2 points of distance PDA plate center 1.5cm, then is placed in 28 DEG C of insulating boxs and cultivates, day by day
Observe bacteriostasis；Above-mentioned opposite culture mode sets 3 repetitions, to be only inoculated with the plate of disease fungus as control.
6, shaken cultivation bacillus subtilis (Bacillus subtilis) K13 in LB liquid medium, obtains withered grass
Bacillus (Bacillus subtilis) K13 strain.
Fig. 1 is plate face-off antagonism of bacillus subtilis (Bacillus subtilis) K13 to anthracnose of rubber trees bacterium
Action diagram, wherein Fig. 1 (a) is the result of bacillus subtilis (Bacillus subtilis) K13 and anthrax-bacilus RC178 face-off
1, Fig. 1 (b) result 2 to stand facing each other for bacillus subtilis (Bacillus subtilis) K13 and anthrax-bacilus RC178, Fig. 1 (c) are
The result 3 of bacillus subtilis (Bacillus subtilis) K13 and anthrax-bacilus RC178 face-off, Fig. 1 (d) are control group ---
The cultivation results of anthrax-bacilus RC178.
The Physiology and biochemistry of 2 bacillus subtilis of embodiment (Bacillus subtilis) K13 is identified
1, V-P is tested
1) principle
For certain bacteriums during glycometabolism, decomposition glucose generates pyruvic acid, and pyruvate decarboxylation generates acetonyl first
Alcohol, acetyl methyl carbinol are oxidized to diacetyl by the oxygen in air in alkaline environment, so with it is smart in peptone in culture medium
Guanidine radicals contained by propylhomoserin works, and generates red compound, then tests for V-P positive.If guanidine radicals content is less in culture medium,
It can be added on a small quantity containing guanidine compound, such as creatine or creatinine.Alpha-Naphthol is added when test can accelerate this reaction.
2) culture medium
5.0 grams of peptone, 5.0 grams of glucose, K2HPO45.0 grams of (or NaCl), 1000 milliliters of distilled water, pH be 7.0~
7.2, it is sub-packed in test tube, every 5 milliliters of test tube, 121 DEG C sterilize 20 minutes.
3) reagent
How phenol alcoholic solution is solution A to 6% α-, and 40% potassium hydroxide is second liquid.
4) method
With agar culture by tested microbionation after glucose peptone water culture, in 28 DEG C of culture 48-96h；In 2
1 milliliter of solution A and 0.4 milliliter of second liquid, shake mixing are added in milliliter culture solution.Strong positive person after five minutes, can produce when test
Pink colour response, it is such as reactionless, culture 4h at 36 ± 1 DEG C should be placed on and observed again.(it is reactionless for a long time, room temperature can be placed in
Overnight), next day constant person is feminine gender.
5) test result
Table 1 is V-P test result.
Table 1 is V-P test result
Obtaining bacillus subtilis (Bacillus subtilis) K13 by experimental result is that V-P test is positive, it is known that withered grass
Bacillus (Bacillus subtilis) K13 contains pyruvate decarboxylase, the acetone that bacterial decomposition glucose can be generated
Acid decarboxylation, generates neutral acetyl methyl carbinol, and the latter can be oxidized generation diacetyl under alkaline environment, diacetyl again with contain
There is the compound of guanidine radicals to react, generate red compound, as V-P experiment is positive.
2, methyl red test
1) principle:
Methyl red test be according to each Pseudomonas of enterobacteriaceae can glucose fermentation, generate third during decomposition glucose
Ketone acid further in decomposition, since the approach of glycometabolism is different, can produce lactic acid, succinic acid, acetic acid and formic acid etc. are a large amount of acid
Product, can make the pH value of culture medium be down to 4.5 hereinafter, make methyl red indicator redden this principle progress test.
2) culture medium
5.0 grams of peptone, 5.0 grams of glucose, 5.0 grams of sodium chloride, 1000 milliliters of distilled water, pH is 7.0~7.2, packing
In test tube, 121 DEG C sterilize 20 minutes.
3) reagent
0.1 gram of methyl red, 300 milliliters of 95% ethyl alcohol, 200 milliliters of distilled water.
4) method
It takes 10 microlitres of K13 fresh seeds liquid to be inoculated in test tube, is placed in 28 DEG C of constant temperature incubations 2,4,6d taking-up, is then training
A drop methyl red reagent is added in nutrient solution, red indicates that methyl red test is positive reaction, and yellow is negative reaction.
5) test result
Table 2 is methyl red test result
2 methyl red test result of table
Obtaining bacillus subtilis (Bacillus subtilis) K13 by experimental result is that methyl red test is positive, it is known that withered
Careless bacillus (Bacillus subtilis) K13 can produce a large amount of acid products.
3, catalase test
1) principle
Bacterium with catalase, energy catalyzing hydrogen peroxide generate water and nascent oxygen, then form molecular oxygen and go out
Existing bubble, is generally used to the type of discriminating bacteria.In gram-positive cocci, staphylococcus and micrococcus luteus generate hydrogen peroxide
Enzyme, and streptococcus is feminine gender, so test is usually used in the preliminary of gram-positive cocci and divides group.
2) method
Slot is taken to be placed in clean test tube or on slide, addend drips 3% hydrogenperoxide steam generator；Or 3% peroxide is directly added dropwise
Change hydrogen solution in the bacterial cultures without blood, observes result immediately.There are a large amount of bubble producers for the positive, does not generate gas
Bubble person is feminine gender.
3) reagent
3% hydrogenperoxide steam generator.
4) test result
Table 3 is catalase test result.
3 catalase test result of table
Obtaining bacillus subtilis (Bacillus subtilis) K13 by experimental result is gram-positive bacteria, is to have
The bacterium of hydrogen oxide enzyme, energy catalyzing hydrogen peroxide generate water and atomic oxygen, then form oxygen molecule, bubble occur.
Embodiment 5
The drafting of bacillus subtilis (Bacillus subtilis) K13 strain growth curve
1) method
Using colorimetric method for determining: LB liquid medium is fitted into 2000 milliliters of triangular flasks, after sterilization and cooling, access activation
Bacillus subtilis (Bacillus subtilis) K13 bacterial strain, be placed in 28 DEG C, cultivate on 180 rpms of shaking table.Often
It is sampled every two h, measures the absorbance (OD600) of 600 nanometers bacterium solutions.
2) result:
Fig. 2 is the growth curve chart of bacillus subtilis (Bacillus subtilis) K13 bacterial strain, and wherein OD600 is vertical
Coordinate, incubation time are abscissa.Because absorbance is determined by bacterium solution density, therefore can be speculated carefully by the variation tendency of absorbance
The growth tendency of bacterium.
As shown in Figure 2: 0~7h, bacillus subtilis (Bacillus subtilis) K13 strain growth is slow, in stopping
Stagnant growth period；7~16h, bacillus subtilis (Bacillus subtilis) K13 strain growth is rapid, is in logarithmic growth
Phase；16~56h, bacillus subtilis (Bacillus subtilis) K13 strain growth is relatively gentle, in stablizing growth period；
And after cultivating 56h, bacillus subtilis (Bacillus subtilis) K13 strain growth starts negative growth occur, is in
The decline phase of growth.
Embodiment 6
The fungistatic effect of bacillus subtilis (Bacillus subtilis) K13 is studied
1) material
Potato dextrose agar: 200 grams of potato, 20 grams of glucose, 20 grams of agar, deionized water is added to
1000 milliliters, steam sterilizing 20 minutes under high pressure.
2) method
A, it screens for the first time: using four-point method.In the potato dextrose agar plate culture medium central got ready in advance, inoculation
Diameter is the anthrax-bacilus RC178 bacteria cake of 6mm, while in symmetrical four away from potato dextrose agar plate culture medium central 25mm
Single bacterial strain is inoculated at point, every plant of bacterium is repeated 3 times, and culture is inverted at 28 DEG C, whether there is or not antibacterial bands to be formed for observation after 3d.
B, secondary screening: method of scoring is used.In the potato dextrose agar plate culture medium central got ready in advance, it is inoculated with diameter
For the anthrax-bacilus RC178 bacteria cake of 6mm, while away from the two sides at potato dextrose agar plate culture medium central 25mm, use
Transfer needle dips the straight line that bacterium to be measured distinguishes the long 30mm of standardized item, and each bacterial strain is repeated 3 times, and measures after culture 4d is inverted at 28 DEG C
The width of each antibacterial band of bacterial strain.(actinomyces growth is slower, need to shift to an earlier date 3d scribing line, than pathogen to ensure that it is colonized).
3) result
The result that the fungistatic effect that table 4 is bacillus subtilis (Bacillus subtilis) K13 is studied.
The fungistatic effect result of study of 4 bacillus subtilis of table (Bacillus subtilis) K13
| Bacterial strain | 1 | 2 | 3 |
| Bacteriostatic diameter (mm) | 6.5 | 5.5 | 6.5 |
Known to.The fungistatic effect of bacillus subtilis (Bacillus subtilis) K13 is very good.
Embodiment 7
Study of Sensitivity of bacillus subtilis (Bacillus subtilis) K13 to antibiotic
1) culture medium
LB solid medium.
2) reagent
Ampicillin, kanamycins, chloramphenicol, streptomysin, tetracycline, erythromycin, rifampin, cephalosporin.
3) method
With filter paper enzyme measurement bacillus subtilis (Bacillus subtilis) K13 to do not have to antibiotic sensibility,
The filter paper of diameter 7mm, high-temperature sterilization are prepared with punch；It is cooled in 50-55 DEG C of LB solid medium and adds after dissolving
Enter 1 milliliter of bacillus subtilis (Bacillus subtilis) K13 seed liquor, rear inverted plate is shaken up, by the filter paper after sterilizing
Be placed in plate, then add antibiotic solution on filter paper, additive amount be equivalent to 50 micrograms per millilitre of ampicillin Amp,
50 micrograms per millilitre of kanamycins Kana, 50 micrograms per millilitre of chloramphenicol Chl, 50 micrograms per millilitre of streptomysin Str, Fourth Ring
Plain 50 micrograms per millilitre of Tet, 50 micrograms per millilitre of Erythromycin E ry, 50 micrograms per millilitre of rifampin Rif, cephamycin C ef
50 micrograms per millilitres are placed in culture in 28 DEG C of constant incubators, respectively at for 24 hours, 48h observe result.
4) result
Table 5 is result of bacillus subtilis (Bacillus subtilis) K13 to the Study of Sensitivity of antibiotic.
Study of Sensitivity result table of 5 bacillus subtilis of table (Bacillus subtilis) K13 to antibiotic
| Antibiotic | 1 | 2 | 3 |
| Cef | 2.8/2.7 | 2.1/2.2 | 2.6/2.7 |
| Amp | — | — | — |
| Kana | 2.5/2.2 | 1.8/1.8 | 2.0/2.0 |
| Chl | — | — | — |
| Str | 1.8/1.8 | 1.7/1.7 | 1.8/1.8 |
| Tet | 2.5/2.4 | 2.3/2.6 | 3.3/3.5 |
| Rif | 1.1/1.1 | 1.1/1.1 | — |
| Ery | 2.0/2.2 | 1.6/1.7 | 3.0/3.5 |
This experiment is quick to various antibiotic mainly for test bacillus subtilis (Bacillus subtilis) K13
Perception, the results showed that ampicillin, chloramphenicol are to the equal unrestraint of bacillus subtilis (Bacillus subtilis) K13.
Bacillus subtilis (Bacillus subtilis) K13 bacterial strain of the invention can effectively inhibit rubber tree anthrax
The growth of germ, to person poultry harmless, green safe, environment compatibility is good, is not easy to produce resistance, and is able to carry out business development, answers
With having a extensive future.
Although having been presented for some embodiments of the present invention herein, it will be appreciated by those of skill in the art that
Without departing from the spirit of the invention, the embodiments herein can be changed.Above-described embodiment is only exemplary, no
It should be using the embodiments herein as the restriction of interest field of the present invention.
Claims (6)
- Bacillus subtilis 1. (Bacillus subtilis) K13, deposit number are as follows: CGMCC No.10917, in 2015 May 27 was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address are as follows: Beijing's southern exposure The institute 3 of area North Star West Road 1.
- 2. bacillus subtilis (Bacillus subtilis) K13 as described in claim 1, which is characterized in that the withered bud Spore bacillus K13 is isolated from rubber leaf.
- The cultural method of bacillus subtilis described in claims 1 or 2 any one 3. (Bacillus subtilis) K13, It is characterized by comprising the following steps:The bacillus subtilis is inoculated in shaken cultivation in LB liquid medium to obtain；The formula of the LB liquid medium: contain in every 1000mL water: tryptone 10g, yeast extract 5g, NaCl 5g； PH is 7.4.
- 4. the application of bacillus subtilis described in claims 1 or 2 any one (Bacillus subtilis) K13, special Sign is, K13 is used to prepare the bacillus subtilis (Bacillus subtilis) has antagonism work to anthracnose of rubber trees bacterium Biochemical reagents.
- 5. the application of bacillus subtilis described in claims 1 or 2 any one (Bacillus subtilis) K13, special Sign is, is used to prepare the biochemical reagents for having inhibition, teratogenesis to anthracnose of rubber trees bacterium mycelia.
- 6. the application of bacillus subtilis described in claims 1 or 2 any one (Bacillus subtilis) K13, special Sign is, is used to prepare the biochemical reagents for having inhibiting effect to anthracnose of rubber trees bacterium mycelia conidia germination.
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