CN106190892B - One bacillus subtilis strain and its application - Google Patents
One bacillus subtilis strain and its application Download PDFInfo
- Publication number
- CN106190892B CN106190892B CN201610541762.1A CN201610541762A CN106190892B CN 106190892 B CN106190892 B CN 106190892B CN 201610541762 A CN201610541762 A CN 201610541762A CN 106190892 B CN106190892 B CN 106190892B
- Authority
- CN
- China
- Prior art keywords
- bacillus subtilis
- ibfcbf
- strain
- bacterial strain
- bacterium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/02—Separating microorganisms from their culture media
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/20—Bacteria; Culture media therefor
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Virology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Agronomy & Crop Science (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Dentistry (AREA)
- Environmental Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The present invention relates to a bacillus subtilis strain and applications, the bacillus subtilis strain is bacillus subtilis IBFCBF-4, and the bacillus subtilis IBFCBF-4 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC No.11233.Bacillus subtilis strain IBFCBF-4 provided by the invention wide, significant characteristic of antagonistic effect with antimycotic range, thus there is good industrial applications prospect.Meanwhile bacillus subtilis strain IBFCBF-4 provided by the invention has good growth-promoting effect to crops.
Description
Technical field
The present invention relates to bacterial strain Cultivating techniques fields, more specifically, be related to a bacillus subtilis strain and its
Using.
Background technique
Soil-borne disease refers to that pathogen such as fungi, bacterium, nematode and virus are lived in the soil with invalid body, and condition is suitable for
When from crop root or stem infringement crop caused by disease.Including standing, withered, line is withered, blueness is withered, withered, epidemic disease, dampings off, root
The soil-borne disease of numerous plants such as rotten, soft corruption, root-knot nematode, cyst nematode.These diseases usually infect plant root or stem,
It falls ill so as to cause root of the crop stem or even complete stool, causes heavy economic losses.
Prevention and control are presently mainly carried out to soil-borne disease by chemical agent, and the long-time service of chemical agent will cause
Water source soil pollution, the ecological balance are destroyed, pesticide residue, Minor diseases are rampant and sex pheromone is made to develop drug resistance etc. one
The serious consequence of series.With the enhancing of people's environmental consciousness, the attention of food-safe problem, and ecological environment is built
If protecting the requirement of bio-diversity and agricultural sustainable development, so that biological prevention and control soil-borne disease becomes current research and opens
The hot spot of hair.
Bacillus (Bacillus sp.) is a kind of sporiferous gram-positive bacterium, and aerobic or amphimicrobian is raw
It is living, the endospore of heat-resisting, drought-enduring, uvioresistant and anti-organic solvent can be generated, produced gemma can be made into pulvis, wettable
The various dosage forms such as pulvis, and rear non-inactivation mixed with chemical pesticide, are ideal screening of biocontrol agents objects.In bacillus,
Bacillus subtilis (Bacillus subtilis) is a kind of with the active bacterium of broad-spectrum antibacterial, bacillus subtilis tool
There is stronger metabolite to generate ability, a variety of antibacterial substances can be generated, thus with good application prospect.
Summary of the invention
Mesh of the invention is to be to provide a bacillus subtilis IBFCBF-4 and its answering in prevention and treatment fungal disease
With plant soil-borne nosomycosis can be effectively prevented using bacillus subtilis provided by the invention and its fermentation liquid produced
Evil.
In order to solve the above technical problem, the present invention provides following technical solutions:
One bacillus subtilis strain, the bacillus subtilis strain are bacillus subtilis IBFCBF-4, and institute
It states bacillus subtilis IBFCBF-4 and is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation is compiled
Number be CGMCC No.11233.
The bacterium colony of the bacillus subtilis IBFCBF-4 is opaque in yellowish, and round or irregular shape, edge be not whole
Together, rough surface fold.
The preparation method of the bacillus subtilis IBFCBF-4, comprising the following steps:
Step 1, soil sample is acquired, soil sample requires salt-soda soil pH value 9.5~10.5, saline and alkaline 0.60~0.70%；
Step 2, soil sample is made to the Soil Slurry of various concentration；
Step 3, the Soil Slurry is added on NA culture medium flat plate and carries out coating process, culture obtains bacterium colony；
Step 4, it selects the different single colonie of form and carries out scribing line conservation, culture obtains separation bacterium；
Step 5, pathogen fungus block is inoculated into the center of PDA culture medium plate, is inoculated with away from plate center equidistant
The separation bacterium selects inhibition zone maximum for bacillus subtilis IBFCBF-4 after culture.
The pathogen fungus block is Oil Tea Anthracnose opportunistic pathogen fungus block.
In a specific embodiment, step 3 cultivation temperature is 28~32 DEG C, and step 5 cultivation temperature is 23~27 DEG C.
The application of the bacillus subtilis IBFCBF-4, the bacillus subtilis IBFCBF-4 and the withered grass bud
The fermentation liquid of spore bacillus IBFCBF-4 is applied to prevention and treatment fungal disease.
Further, the fungal disease is that soil passes fungus diseases.Preferably, it is that Oil Tea Anthracnose is former that the soil, which passes fungus diseases,
Bacterium, Rhizoctonia solani Kuhn, flax anthrax-bacilus, sharp fusarium flax specialized form, phytophthora blight of pepper, tomato gray mould bacterium, Fusarium oxysporum, standing grain
Paddy sickle-like bacteria, fusarium moniliforme, Rhizoctonia cerealis, early epidemic germ, gaeumannomyce bacterium or sclerotinite.
In a specific embodiment, it is described solution bacillus subtilis IBFCBF-4 fermentation liquid preparation include: will be withered
Careless bacillus IBFCBF-4 is seeded in liquid fermentation medium, and in 25-30 DEG C of progress fermented and cultured, shaking table shakes 2-4 days,
Obtain the fermentation liquid of bacillus subtilis IBFCBF-4.
In a specific embodiment, the liquid fermentation medium includes following components: 8~12g/L according to concentration
Tryptone, the powdered beef of 2~4g/L, the glucose of 15~25g/L, the NaCl of 4~6g/L, the pH of liquid fermentation medium
For 7.2-7.4.
Bacillus subtilis strain IBFCBF-4 provided by the invention is wide with antimycotic range, antagonistic effect is significantly special
Property, thus there is good industrial applications prospect.Meanwhile bacillus subtilis strain IBFCBF-4 provided by the invention is to agriculture
Crop has good growth-promoting effect.
Especially, IBFCBF-4 significantly inhibits Oil Tea Anthracnose opportunistic pathogen, and field trial shows IBFCBF-
The control efficiency of 4 pairs of oil tea anthraxs is significantly higher than IBFCBF-1.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, those of ordinary skill in the art are come
It says, without creative efforts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is the preparation flow figure of bacillus subtilis IBFCBF-4 provided in an embodiment of the present invention；
Fig. 2 is the colonial morphology figure of bacillus subtilis IBFCBF-4 provided in an embodiment of the present invention；
Fig. 3 is the Gram's staining figure of bacillus subtilis IBFCBF-4 provided in an embodiment of the present invention；
Fig. 4 is the spore staining figure of bacillus subtilis IBFCBF-4 provided in an embodiment of the present invention；
Fig. 5 is the PCR primer agarose electrophoresis figure of bacillus subtilis IBFCBF-4 provided in an embodiment of the present invention；
Fig. 6 is the phylogenetic tree of the 16S sequence construct of bacillus subtilis IBFCBF-4 provided in an embodiment of the present invention
Figure；
Fig. 7 is bacillus subtilis IBFCBF-4 provided in an embodiment of the present invention and IBFCBF-1 plate antagonism oil tea anthrax
Pathogen contrast schematic diagram；
Fig. 8 is the schematic diagram of bacillus subtilis IBFCBF-4 prevention and treatment capsicum epidemic disease provided in an embodiment of the present invention.
Specific embodiment
Bacillus subtilis strain provided in an embodiment of the present invention makes bacillus subtilis provided by the invention and its gives birth to
The fermentation liquid of production can effectively prevent plant silborne fungal diseases.
Technical solution in embodiment in order to enable those skilled in the art to better understand the present invention, and make of the invention real
The above objects, features, and advantages for applying example can be more obvious and easy to understand, with reference to the accompanying drawing to the technology in the embodiment of the present invention
Scheme is described in further detail.
Bacillus subtilis strain provided in an embodiment of the present invention is bacillus subtilis IBFCBF-4, the withered grass gemma
Bacillus IBFCBF-4 was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on August 11st, 2015
(abbreviation CGMCC, depositary institution address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica；
Postcode: 100101), deposit number is CGMCC No.11233.
Attached drawing 1 is please referred to, attached drawing 1 shows the preparation stream of bacillus subtilis IBFCBF-4 provided in an embodiment of the present invention
Cheng Tu.The preparation step of bacillus subtilis IBFCBF-4 provided in an embodiment of the present invention are as follows:
S01: soil sample, and collected soil sample is finely ground, the soil sample in the embodiment of the present invention are acquired by multi-point sampling method
It is derived from Changling, salt-soda soil pH value 10.0, saline and alkaline 0.65%.；
S02: finely ground soil sample being put into the centrifuge tube equipped with sterile water and is fullyd shake, and it is 10 that concentration, which is made,-1Soil
Suspension；
S03: by the Soil Slurry gradient dilution, the Soil Slurry of various concentration is obtained；
S04: choosing concentration is 10-5、10-6With 10-7Soil Slurry, and be added on NA culture medium flat plate and applied
Cloth processing, is put into 30 DEG C of incubator and cultivates, obtain bacterium colony；
S05: selecting the different single colonie of form and carry out scribing line conservation, cultivate 24 hours, separation bacterium is obtained, by the separation
Bacterium is placed in 4 DEG C of refrigerator and saves backup；
S06: flax is found into withered, capsicum epidemic disease enzyme with the punch of diameter 0.5cm, Oil Tea Anthracnose opportunistic pathogen fungus block is inoculated with respectively
In different PDA culture medium plate centers (it is as a comparison that flax, which founds withered, capsicum epidemic disease enzyme fungus block), in anomaly plate center 2.5cm
The different separation bacterium of equidistant inoculation at right-angled intersection, not to be inoculated with separation bacterium as control (CK), 3 repetitions of progress per treatment
Experiment cultivates under the conditions of temperature is 25 DEG C, when control group (CK) covers with whole vessel, records the size of inhibition zone, selects
Maximum to Oil Tea Anthracnose opportunistic pathogen inhibition zone is bacillus subtilis IBFCBF-4.
Wherein, NA culture medium is nutrient agar, and the composition of the nutrient agar includes 10g/L according to concentration
Tryptone, the powdered beef of 3g/L, the agar of the NaCl of 5g/L, 20g/L.The pH value of NA culture medium in the preparation be 7.2~
7.4, and by the sterilization treatment of 20min, and temperature when sterilizing is 121 DEG C.PDA culture medium is potato dextrose agar training
Support base.
The embodiment of the invention provides bacillus subtilis IBFCBF-4 and IBFCBF-1 (number of patent application
201510867702.4) contrast schematic diagram of antagonism Oil Tea Anthracnose opportunistic pathogen, schematic diagram please refer to (the left side IBFCBF- of attached drawing 7
4).From attached drawing 7, it can be seen that when control group all covers with vessel, it is inoculated with bacillus subtilis IBFCBF-4 separation bacterium
PDA culture medium on there is apparent inhibition zone, and the diameter of inhibition zone, in 2.0cm or more, inhibition zone size is significant
Higher than IBFCBF-1 to the inhibition zone size 1.2cm of oil tea anthrax.It can illustrate that the embodiment of the present invention mentions by the appearance of inhibition zone
The bacillus subtilis IBFCBF-4 of confession significantly inhibits Oil Tea Anthracnose opportunistic pathogen, it is thus possible to application well
In prevention and treatment fungal disease, field trial shows that IBFCBF-4 up to 74.40%, is significantly higher than the control efficiency of oil tea anthrax
The control efficiency 40.69% of IBFCBF-1.
A situation arises for Oil Tea Anthracnose after table 1:IBFCBF-1 and IBFCBF-4 processing
| Processing | Disease index before being administered | 14d disease index after application | Control efficiency (%) |
| Clear water control | 20.63±1.45a | 38.52±2.42a | — |
| IBFCBF-1 | 22.13±1.33a | 32.74±2.27b | 40.69±4.69b |
| IBFCBF-4 | 19.27±0.88a | 23.85±2.18c | 74.40±2.23a |
Wherein control efficiency %=[(disease index after disease index-control group application before control group is administered)-(processing group
Disease index after disease index-processing group application before being administered)]/(control group is administered the state of an illness after preceding disease index-control group application
Index) × 100.
The preparation of the fermentation liquid of bacillus subtilis IBFCBF-4 provided in an embodiment of the present invention includes: by bacillus subtilis
Bacterium IBFCBF-4 is seeded in liquid fermentation medium, and in 25-30 DEG C of progress fermented and cultured, shaking table shakes 2-4 days, obtains withered grass
The fermentation liquid of bacillus IBFCBF-4.
Wherein, the revolving speed of shaking table concussion is 180r/min.
Liquid fermentation medium includes following components: the tryptone of 10g/L, the powdered beef of 3g/L, 20g/L according to concentration
Glucose, the NaCl of 5g/L.The pH value of liquid fermentation medium in the preparation is 7.2~7.4, and by the sterilizing of 20min
Processing, and temperature when sterilizing is 121 DEG C.
The present invention is identified that bacterial strain provided in an embodiment of the present invention, the identification includes the following contents:
1, Morphological Identification
Bacterial strain provided in an embodiment of the present invention is crossed on NA culture medium flat plate, is then reversed plate, is in temperature
The growing state for observing and recording bacterium colony on plate for 24 hours is cultivated under conditions of 30 DEG C.The bacterium of bacterial strain provided in an embodiment of the present invention
It falls aspect graph and please refers to attached drawing 2.
It is opaque, it can be seen that the bacterium colony of bacterial strain provided in an embodiment of the present invention is in yellowish from attached drawing 2, it is round or
Irregular shape, edge is irregular, rough surface fold.
Further, Gram's staining and spore staining carried out to bacterial strain provided in an embodiment of the present invention with kit, and
It oily microscopic observation bacterial strain and takes pictures to bacterial strain.The Gram's staining of the bacterial strain and spore staining please refer to attached drawing 3 and attached drawing
4。
From attached drawing 3, it can be seen that after Gram's staining, bacterial strain provided in an embodiment of the present invention is rod-shaped, and in indigo plant
Purple is gram-positive bacteria.From attached drawing 4, it can be seen that after spore staining, bacterial strain thallus provided in an embodiment of the present invention
Aobvious blue, the aobvious red of gemma, thus illustrates that bacterial strain provided by the invention can generate gemma.
2, Physiology and biochemistry is identified
(1) catalase is tested
3% hydrogen peroxide is directly added dropwise in the liquid medium of bacterial strain, observes immediately.If there is a large amount of bubbles to generate,
It is then the positive；If not generating bubble, for feminine gender.Bacterial strain provided by the invention generates a large amount of bubbles immediately, and experimental result is sun
Property.
(2) oxidizing ferment is tested
One jiao of extracting waste cleaning filter paper dips a small amount of bacterial strain bacterium colony, and the hydrochloride base that concentration is 1% is added to benzene two
Amine aqueous solution one drips, positive pinkiness immediately, and color can gradually deepen.In this experiment, bacterium colony pinkiness, face
Color is gradually deepened, and experimental result is the positive.
(3) Starch Hydrolysis is tested
The strain point of bacterial strain is connected on starch culture-medium, is cultivated for 24 hours under conditions of temperature is 37 DEG C, a small amount of iodine is added dropwise
Liquid gently rotates on starch culture-medium plate, is evenly distributed on iodine solution on starch culture-medium plate, observes periphery of bacterial colonies
Situation.If colorless and transparent circle, which occurs, in periphery of bacterial colonies shows the ability for having hydrolysis starch, conversely, not having.The week of this bacterial strain bacterium colony
It is with transparent circle generation, the ability that there is hydrolysis starch thus, it is possible to illustrate this bacterial strain.
(4) methyl red MR is tested
This small amount of bacterial strain of picking, and be inoculated on collective media, it is cultivated 3~5 days under conditions of temperature is 30 DEG C,
Culture solution 1ml is taken after culture, and the drop of methyl red indicator 1~2 is added, and the positive is in cerise, and weakly positive is in pale red, yin
Property is yellow.In this experiment, bacterium solution turns yellow, so for feminine gender.
(5) VP is tested
This small amount of bacterial strain of picking, and it is inoculated in collective media, it is cultivated 4 days under conditions of temperature is 30 DEG C, culture
After take culture solution 2.5ml that a naphthols absolute alcohol solution 0.6ml is first added, add concentration be 40% potassium hydroxide it is water-soluble
Liquid 0.2ml shakes 2-5min, and red is usually presented in positive bacteria immediately, if redfree occurs, is statically placed in the constant temperature of room temperature or 30 DEG C
In case, if not showing red in 2h still, it can determine that as feminine gender.In this experiment, bacterium solution reddens immediately, so for the positive.
(6) gelatin liquefaction is tested
This bacterial strain is taken, percutaneous puncture-inoculation is located at this bacterial strain at the 2/3 of gelatin depth in gelatin.It is 20 DEG C in temperature
Under conditions of cultivate 5-7d.Whether observation this bacterial strain of result is liquefied by bacterium daily, positive for test if being liquefied；If no
It is liquefied, is then feminine gender.In this experiment, gelatin is liquefied, therefore this bacterial strain is the positive.
(7) nitrate reduction is tested
By this strain inoculated in nitrate broth, then shaking table culture 3d under conditions of temperature is 28 DEG C takes 5mL to train
Nutrient solution illustrates that color developing agent is added by kit (Hai Bo Bioisystech Co., Ltd nitrate reduction kit), turned yellow as the positive,
Conversely, not changing color as feminine gender.In this experiment, bacterium solution turns yellow, and illustrates this bacterial strain for the positive.
(8) hydrogen sulfide experiment is produced
By this bacterial strain percutaneous puncture-inoculation in lead acetate medium, 24~48h is cultivated under conditions of temperature is 35 DEG C, and see
Examine result.If culture medium blackening, for the positive；Not blackening is then feminine gender.In this experiment, culture medium discoloration illustrates this bacterium
Strain is the positive.
(9) citrate utilizes experiment
The streak inoculation on simon Si Shi citrate medium inclined-plane of this bacterial strain is chosen, is trained under conditions of temperature is 37 DEG C
It supports 3-7 days.If culture medium is alkalinity person, i.e., indicator blue or pink are the positive；If culture medium is non-discolouring, for yin
Property.In this experiment, culture medium does not change colour, illustrates this bacterial strain for feminine gender.
(10) lecithin activity is tested
The surface of Fresh Egg is carried out disinfection with 75% ethyl alcohol, and egg is beaten into a hole with the tweezers of sterilizing, is inclined
Then yolk is sucked out with aseptic straw in egg white, be added in the NA culture medium for being cooled to 50 DEG C or so after melting, after being mixed evenly
Fell plate, and point connects this bacterial strain, cultivates for 24 hours under conditions of temperature is 30 DEG C, and observed.If colony edge occurs muddy
Circle person is enzyme positive.In this experiment, there is apparent muddy circle in colony edge, illustrates this bacterial strain for the positive.
(11) malonate utilizes experiment
Picking culture 12h lawn is inoculated in malonate culture medium, cultivates 24-48h under conditions of temperature is 35 DEG C, trains
Base is supported by green change indigo plant person as the positive, otherwise is feminine gender.In this experiment, culture medium discoloration illustrates this bacterial strain for the positive.
(12) glucose fermentation is tested
A small amount of this bacterial strain percutaneous puncture-inoculation of picking is cultivated under conditions of temperature is 30 DEG C in glucose oxidation-fermentation medium
3d observes the variation of culture medium color.If needing to continue to observe 7d without color change, culture medium flavescence person is fermented type.?
This time in experiment, culture medium turns yellow, and illustrates this bacterial strain for the positive.
(13) cellulose decomposition is tested
When having obvious bacterium colony, a drop is instilled into plate on sodium carboxymethylcellulose solid medium for spread plate
Congo red dye liquor is managed, and is evenly distributed in Congo red dye liquor on plate.1ml sodium chloride solution is added after 15 minutes, impregnates
After 15 minutes, dyeing is washed away, sees whether to generate transparent circle.If there is transparent circle generation, for the positive.In this experiment, have
Transparent circle generates, and illustrates this bacterial strain for the positive.
(14) galactose utilization experiment is positive
By this strain inoculated in gala sugar culture-medium, 2d, observation bacterium colony growth are cultivated under conditions of temperature is 30 DEG C
Situation can use galactolipin if bacterium colony is formed, conversely, not all right.In this experiment, thalli growth illustrates that this bacterial strain can
To utilize galactolipin.
(15) arabinose utilizes experiment
By this strain inoculated in arabinose culture medium, 2d is cultivated under conditions of temperature is 30 DEG C, observation bacterium colony is raw
Long situation can use arabinose if bacterium colony is formed, conversely, not all right.In this experiment, thallus is not grown, and illustrates this
Bacterial strain cannot utilize arabinose.
(16) mannose utilizes experiment
By this strain inoculated in sweet dew sugar culture-medium, 2d, observation bacterium colony growth are cultivated under conditions of temperature is 30 DEG C
Situation can use mannose if bacterium colony is formed, conversely, not all right.In this experiment, thalli growth, this bacterial strain can benefit
Use mannose.
(17) D-Fructose utilizes experiment
By this strain inoculated in D-Fructose culture medium, 2d, observation bacterium colony growth are cultivated under conditions of temperature is 30 DEG C
Situation can use D-Fructose if bacterium colony is formed, conversely, not all right.In this experiment, thalli growth, this bacterial strain can benefit
Use D-Fructose.
(18) D- xylose utilization is tested
By this strain inoculated in D- xylose media, 2d, observation bacterium colony growth are cultivated under conditions of temperature is 30 DEG C
Situation can use D- xylose if bacterium colony is formed, conversely, not all right.In this experiment, there is bacterium colony to be formed, this bacterial strain can be with
Utilize D- xylose.
To sum up, Physiology and biochemistry qualification result such as table 2.
Table 2: the Physiology and biochemistry qualification result of this bacterial strain
| Table feature | Response feature | Table feature | Response feature |
| Contact enzymatic determination | + | Citrate utilizes | + |
| Oxidase assay | + | Lecithin activity measurement | + |
| Starch Hydrolysis measurement | + | Malonate utilizes | + |
| Methyl red MR measurement | — | Cellulose decomposition | + |
| VP experiment | + | Galactose utilization | + |
| Gelatin liquefaction measurement | + | Arabinose utilizes | — |
| Nitrate reduction measurement | + | Mannose utilizes | + |
| Glucose fermentation | + | D-Fructose utilizes | + |
| Produce hydrogen sulfide measurement | — | D- xylose utilization | + |
Wherein ,+be expressed as this bacterial strain and have reaction or can use ,-be expressed as this bacterial strain and do not react or cannot utilize.
3,16S rDNA sequence is analyzed
The embodiment of the present invention uses health to extract in this bacterial strain for century Biotechnology Co., Ltd's genome extraction kit
DNA.
Wherein, said extracted kit includes the PCR reaction system of 50 μ L, and PCR reaction system include 25 μ L 2 ×
Master Mix；The upstream primer of 2.5 μ L；The downstream primer of 2.5 μ L；The dd H2O of 18 μ L；The template DNA of 2 μ L.
PCR reaction condition are as follows: temperature be 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 0.5min；53 DEG C of annealing 0.5min；72
DEG C extend 1min；30 circulations, 72 DEG C of extension 5min, 4 DEG C of preservations.
Attached drawing 5 is please referred to, the PCR product that attached drawing 5 shows bacillus IBFCBF-4 provided in an embodiment of the present invention is used
The electrophoretogram of 1% agarose gel electrophoresis detection.PCR product is sequenced by Changsha your Biotechnology Co., Ltd of dimension generation, is surveyed
Sequence result is referring to the nucleotide sequence in subordinate list.Gained sequence is subjected to homology sequence by NCBI-BLAST and compares analysis,
Obtain the higher sequence of similitude.With MEGA6.06 software building phylogenetic tree, the 16S rDNA sequence of this bacterial strain with it is withered
The homology of careless bacillus (Bacillus subtilis) reaches 100%, and the development tree graph of this bacterial strain please refers to attached drawing 6.
In summary morphological observation, Physiology and biochemistry identification and 16S rDNA the sequencing results, can determine this bacterial strain
IBFCBF-4 is bacillus subtilis Cordycepps bacillus (Bacillus subtilis), is named as bacillus subtilis
IBFCBF-4。
Bacillus subtilis IBFCBF-4 and its fermentation liquid provided in an embodiment of the present invention can be applied to prevention and treatment nosomycosis
Evil, the fungal disease are that soil biography fungus diseases are Rhizoctonia solani Kuhn (Rhizoctonia solani), flax anthrax-bacilus
(Colletotrichum linicola), sharp fusarium flax specialized form (Fusarium oxysporum Schltdl.ex
Snyder et Hansen f.sp.lini), phytophthora blight of pepper (Phytophthora capsici), tomato gray mould bacterium
(Botrytis cinerea), Fusarium oxysporum (Fusarium oxysporum), Fusarium graminearum (F.graminearum),
Fusarium moniliforme (F.verticillioides), Rhizoctonia cerealis (R.cerealis), early epidemic germ (Alternaria
Solani), gaeumannomyce bacterium (Gaeumannomyces graminis) or sclerotinite (Sclerotinia sclerotiorum)
Deng soil pass fungus diseases.
The embodiment of the present invention has carried out bacillus subtilis IBFCBF-4 and its fermentation liquid to true by taking phytophthora blight of pepper as an example
The research of fungus diseases, research contents are as follows.
(1) selection of culture medium
The culture of bacillus subtilis IBFCBF-4 uses NB fluid nutrient medium (i.e. beef extract-peptone fluid nutrient medium),
The preparation of the NB fluid nutrient medium includes the peptone of 10g, the powdered beef of 3g and the NaCl of 5g, and above-mentioned substance uses deionized water
It is settled to 1000ml, and adjusting pH value is 7.2-7.4, then 1 × 105Sterilize 20min under the pressure of Pa, and the training of NB liquid is made
Support base.
Capsicum epidemic disease enzymophathy fungal pathogens use PDA liquid medium, and the preparation of the PDA liquid medium includes the peeling of 200g
Potato, the glucose of 20g, above-mentioned substance is settled to 1000mL using deionized water, then 1 × 105It goes out under the pressure of Pa
PDA liquid medium is made in bacterium 20min.
(2) selection of capsicum variety
The Sweet Pepper Varieties eggplant door that capsicum selects Vegetable Research Inst., Hunan Prov. Agriculture Science Academy to provide.By the seed of eggplant door with dense
Degree carries out surface sterilization 20min for 10% hydrogen peroxide, then uses sterile water shower 3 times, and be placed inCulture dish
In, constant temperature half-light vernalization under conditions of temperature is 30 DEG C.The seed for choosing a length of 0.5cm of bud is sowed in 9 hole alms bowls, and matrix is city
Nutrition Soil is sold, capsicum is placed in 30 ± 1 DEG C of phjytotron and is cultivated, and the intensity of illumination at this time is 12000Lux, illumination
Period is L ︰ D=14 ︰ 10, RH=80% ± 10%.
(3) specific experiment designs
Experiment is divided into 3 groups:
CK1:NB culture medium；CK2: clear water；T: bacillus subtilis IBFCBF-4 bacterium solution.
Wherein, bacillus subtilis IBFCBF-4 is 30 DEG C in temperature, and revolving speed cultivates 48h under conditions of being 180r/min,
Then bacterium solution is diluted to 10 with sterile water5CFU/mL.Capsicum elicitin pathogenic bacteria is 30 DEG C in temperature, and revolving speed is 180r/min's
Under the conditions of cultivate 7d, bacterium solution is then diluted to 10 with sterile water5CFU/mL。
In experiment, pepper seed one of every hole sowing budding in 9 hole alms bowls guarantees the survival of 9 alms bowl Hot Pepper Seedlings, and by 9 alms bowls
Hot Pepper Seedling is divided into three groups, every group of three alms bowls, when capsicum was cultivated to the 3-4 leaf phase, applies capsicum elicitin pathogenic bacteria in the rhizosphere of capsicum
Then bacterium solution 2.5mL adds NB culture medium, clear water and the bacillus subtilis of 2.5mL respectively into the young plant for be divided into three groups
IBFCBF-4 bacterium solution.Apply 5mL nutrient solution every the every hole 2d, to guarantee that matrix is more wet, capsicum being capable of normal growth.Observation
Chili growth situation, after 60d to capsicum carry out plant height, stem thickness, internode away from, chlorophyll content in leaf blades, top half fresh weight and dry
The measurement of weight and the growth indexes such as root system fresh weight and dry weight counts every group of capsicum epidemic disease morbidity strain number.
(4) measurement method
From pepper plant base portion to stem, the distance between top trunk diameter growth point is plant height, the nearly root knot of plant when measurement
First segment stem diameter be stem thickness.Random three pieces leaf of often selecting good strains in the field for seed, measures its chlorophyll content with chlorophyll meter.Plant is per small
The length for saving stem is internode away from first three section of nearly root knot is chosen in this experiment.Pepper plant is cut from first node of nearly root knot
It is disconnected, the weight of top half and root system part is weighed respectively, is denoted as fresh weight, is then placed into 85 DEG C of baking oven and is dried to perseverance
Weight, weighs its weight respectively again, is denoted as their dry weight.Experimental result please refers to table 3, table 4 and attached drawing 8.
Influence of 3: the three kinds of different disposal methods of table to chili growth
Table 4: a situation arises for the capsicum epidemic disease handled with three kinds of distinct methods
| Processing | It falls ill strain number (strain) | Disease incidence (%) | Control efficiency (%) |
| T | 1.67±0.33b | 18.52±3.7b | 64.45±2.22a |
| CK1 | 3.67±0.33a | 40.74±3.7ab | 17.78±9.69a |
| CK2 | 4.67±0.88a | 51.85±9.8a | — |
Wherein, diseased plant rate %=morbidity strain number/investigation total strain number × 100；Control efficiency %=(1- processing group morbidity strain
Rate/control group diseased plant rate) × 100.
From table 2, it can be seen that the processing of T group make capsicum have higher plant height, thicker stem, and second successively away from,
Third successively away from, chlorophyll content, top half fresh weight, top half dry weight, root fresh weight and root dry weight data all than remaining two
The data of group are big.It is thus, provided in an embodiment of the present invention withered meanwhile it can be seen that pepper plant grows fine from attached drawing 8
Careless bacillus IBFCBF-4 and its fermentation liquid can effectively promote the growth of plant.From in table 3, it can be seen that from T group
Reason makes the disease incidence of capsicum significantly lower than other two groups of disease incidence, and control efficiency is even more to be significantly greater than other two groups of prevention and treatment
Effect, thus, it is possible to illustrate that it is significant short of money that bacillus subtilis IBFCBF-4 and its fermentation liquid provided in an embodiment of the present invention have
Anti- effect.
The embodiments of the present invention described above are not intended to limit the scope of the present invention.It is any in the present invention
Spirit and principle within made modifications, equivalent substitutions and improvements etc., should all be included in the protection scope of the present invention.
Claims (3)
1. a bacillus subtilis strain, which is characterized in that the bacillus subtilis strain is bacillus subtilis
IBFCBF-4, and the bacillus subtilis IBFCBF-4 is preserved in China Committee for Culture Collection of Microorganisms's commonly micro- life
Object center, deposit number are CGMCC No.11233.
2. bacillus subtilis strain according to claim 1, which is characterized in that the bacillus subtilis IBFCBF-4
Bacterium colony in yellowish, opaque, round or irregular shape, edge is irregular, rough surface fold.
3. the application of bacillus subtilis IBFCBF-4 according to claim 1 or 2, which is characterized in that the withered grass bud
The application of spore bacillus IBFCBF-4 and its fermentation liquid in prevention and treatment soil biography fungus diseases Oil Tea Anthracnose opportunistic pathogen and phytophthora blight of pepper.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610541762.1A CN106190892B (en) | 2016-07-11 | 2016-07-11 | One bacillus subtilis strain and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610541762.1A CN106190892B (en) | 2016-07-11 | 2016-07-11 | One bacillus subtilis strain and its application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106190892A CN106190892A (en) | 2016-12-07 |
| CN106190892B true CN106190892B (en) | 2019-07-12 |
Family
ID=57476217
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610541762.1A Active CN106190892B (en) | 2016-07-11 | 2016-07-11 | One bacillus subtilis strain and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106190892B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106635929A (en) * | 2017-02-14 | 2017-05-10 | 河南工业大学 | Bacillus subtilis strain and application of antifungal metabolite in grain storage |
| CN107058172B (en) * | 2017-02-27 | 2020-01-03 | 中国农业科学院植物保护研究所 | Bacillus thuringiensis and application thereof |
| CN110819565B (en) * | 2019-11-20 | 2021-04-13 | 青岛农业大学 | Salt-tolerant bacillus BW9 with plant pathogenic bacteria inhibiting effect and application thereof |
| CN112831443B (en) * | 2021-02-22 | 2022-08-02 | 青岛农业大学 | Bacillus subtilis, microbial inoculum and application thereof in preventing and treating plant diseases |
| CN113801804B (en) * | 2021-03-24 | 2022-11-15 | 云南省农业科学院农业环境资源研究所 | Banana wilt biocontrol antagonistic strain and application thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101475922A (en) * | 2009-01-09 | 2009-07-08 | 中南林业科技大学 | Bacillus subtilis Y13 for effectively preventing and treating camellia anthracnose and use thereof |
| CN102154177B (en) * | 2011-01-26 | 2012-08-15 | 中南林业科技大学 | Oil-tea disease-resistant bacterial strain and application thereof |
-
2016
- 2016-07-11 CN CN201610541762.1A patent/CN106190892B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN106190892A (en) | 2016-12-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109055281B (en) | Bacillus belgii ZF2 and application thereof in plant disease control | |
| CN105368747B (en) | One bacillus amyloliquefaciens bacterial strain and its application | |
| CN102433282B (en) | Bacillus subtilis NB12, as well as culture method and application thereof | |
| CN105557756B (en) | Plant bacteriostasis method adopting bacillus methylotrophicus strain NKG-1 | |
| CN106190892B (en) | One bacillus subtilis strain and its application | |
| CN107736379B (en) | Application of bacillus amyloliquefaciens in preventing and treating plant fungal diseases | |
| CN107083349B (en) | The white black streptomycete of one plant of disease prevention growth-promoting and its preparation and application of metabolite | |
| CN102433281B (en) | Streptomyces katrae NB20, as well as culture method and application thereof | |
| CN102676398B (en) | Separation and purification method of endophytic fungi from ginkgo biloba | |
| CN108085269A (en) | Methylotrophic Bacillus strain IBFCBF-2 and its application | |
| CN103013860A (en) | Preparation and application of biological control bacterial strain for diseases of ginseng plant | |
| CN109609402A (en) | Te Jila bacillus Bacillus tequilensis XG18 and application | |
| CN113308392B (en) | Application of Nori endophytic Siamese bacillus | |
| CN107075459A (en) | Novel bacterial of bacillus and application thereof | |
| CN109868236A (en) | One plant of Jie meter La series bacillus, its tunning and preparation method and application | |
| CN103109870B (en) | Application of fermentation supernatant liquid of pseudomonas | |
| CN107699526A (en) | One plant of actinomycetes strain for preventing and treating gray mold and its application | |
| CN101942395B (en) | Aspergillus aculeatus | |
| CN110317747A (en) | A kind of bacillus amyloliquefaciens JT68 and its application in prevention and treatment tea anthracnose | |
| CN109321500A (en) | One bacillus amyloliquefaciens bacterial strain and its application in prevention and treatment Oil Tea Anthracnose evil | |
| CN110184224A (en) | One staphylococcus epidermidis and its application | |
| CN107629985B (en) | Plant endophytic bacterium with antagonistic effect on plant pathogenic fungi | |
| CN105400717A (en) | Bacterial strain HBRM-16 capable of promoting growth of roots of rubber tree and application of bacterial strain HBRM-16 | |
| Lang et al. | Identification of endophytes with biocontrol potential from Ziziphus jujuba and its inhibition effects on Alternaria alternata, the pathogen of jujube shrunken-fruit disease | |
| CN103210961A (en) | Biological pesticide for preventing ginseng gray mold and black spot |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| EE01 | Entry into force of recordation of patent licensing contract | ||
| EE01 | Entry into force of recordation of patent licensing contract |
Application publication date: 20161207 Assignee: Shenzhen Hanzhi Biotechnology Co.,Ltd. Assignor: INSTITUTE OF BAST FIBER CROPS, CHINESE ACADEMY OF AGRICULTURAL SCIENCES Contract record no.: X2022980023911 Denomination of invention: A bacillus subtilis strain and its application Granted publication date: 20190712 License type: Common License Record date: 20221129 |