CN106190892A - One bacillus subtilis strain and application thereof - Google Patents
One bacillus subtilis strain and application thereof Download PDFInfo
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Abstract
The present invention relates to a bacillus subtilis strain and application, described bacillus subtilis strain is bacillus subtilis IBFCBF 4, and described 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.The bacillus subtilis strain IBFCBF 4 that the present invention provides has that antifungal scope is wide, the significant characteristic of antagonistic effect, thus has good industrial applications prospect.Meanwhile, the bacillus subtilis strain IBFCBF 4 that the present invention provides has good growth-promoting effect to crops.
Description
Technical field
The present invention relates to bacterial strain Cultivating techniques field, more specifically, relate to a bacillus subtilis strain and
Application.
Background technology
Soil-borne disease refers to that pathogen such as fungus, antibacterial, nematicide and virus are lived in soil with invalid body, and condition is suitable
Time encroach on crop from crop root or stem and the disease that causes.Including vertical withered, stricture of vagina is withered, blue or green withered, withered, epidemic disease, damping off, root
The soil-borne disease of numerous plants such as corruption rotten, soft, root-knot nematode, Cyst nematode.These diseases generally infect plant root or stem,
Thus cause root of the crop stem or even Herb morbidity, cause heavy economic losses.
It is presently mainly and by chemical agent soil-borne disease is carried out prevention and control, and the life-time service of chemical agent, can cause
Water source soil pollution, ecological balance is destroyed, pesticide residues, Minor diseases rampant and makes sex pheromone develop immunity to drugs etc. one
The consequence that series is serious.Along with the enhancing of people's environmental consciousness, the attention to food-safety problem, and ecological environment is built
If, protection bio-diversity and the requirement of agricultural sustainable development so that biological prevention and control soil-borne disease becomes current research and opens
The focus sent out.
Bacillus cereus (Bacillus sp.) is a sporiferous gram-positive bacterium of class, aerobic or amphimicrobian life
Live, it is possible to producing heat-resisting, drought-enduring, uvioresistant and the endospore of anti-organic solvent, produced spore can be made into powder, wettability
The various dosage form such as powder, and rear non-inactivation mixed with chemical pesticide, be preferable screening of biocontrol agents object.In bacillus cereus,
Bacillus subtilis (Bacillus subtilis) is a kind of antibacterial with broad-spectrum antibacterial activity, and this bacillus subtilis has
Stronger metabolite is had to produce ability, it is possible to produce multiple antibacterial substance, thus have preferable application prospect.
Summary of the invention
The mesh of the present invention is to be to provide a bacillus subtilis IBFCBF-4 and answering in preventing and treating fungal disease thereof
With, plant soil-borne mycosis can effectively be prevented and treated by the bacillus subtilis using present invention offer and the fermentation liquid produced thereof
Evil.
In order to solve above-mentioned technical problem, the present invention provides following technical scheme:
One bacillus subtilis strain, described bacillus subtilis strain is bacillus subtilis IBFCBF-4, and institute
Stating bacillus subtilis IBFCBF-4 and be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation is compiled
Number it is CGMCC No.11233.
The bacterium colony of described bacillus subtilis IBFCBF-4 is slightly yellow, opaque, and circular or irregular shape, edge is the most whole
Together, rough surface fold.
The preparation method of described bacillus subtilis IBFCBF-4, comprises the following steps:
Step 1, gathers soil sample, and soil sample requires salt-soda soil pH value 9.5～10.5, saline and alkaline 0.60～0.70%；
Step 2, makes the Soil Slurry of variable concentrations by soil sample；
Step 3, joins described Soil Slurry and is coated on NA culture medium flat plate processing, cultivate and obtain bacterium colony；
Step 4, selects single bacterium colony that form differs and carries out conservation of ruling, and cultivates and obtains separating bacterium；
Step 5, is inoculated into the center of PDA culture medium flat plate by pathogen truffle, is inoculating away from plate center equidistant
Described separation bacterium, select after cultivation inhibition zone maximum for bacillus subtilis IBFCBF-4.
Described pathogen truffle is Oil Tea Anthracnose former bacterium truffle.
In a detailed description of the invention, step 3 cultivation temperature is 28～32 DEG C, and step 5 cultivation temperature is 23～27 DEG C.
The application of described bacillus subtilis IBFCBF-4, described bacillus subtilis IBFCBF-4 and described hay bud
The fermentation liquid of spore bacillus IBFCBF-4 is applied to prevent and treat fungal disease.
Further, described fungal disease is that soil passes fungus diseases.Preferably, described soil passes fungus diseases is that Oil Tea Anthracnose is former
Bacterium, Rhizoctonia solani Kuhn, Caulis et Folium Lini anthrax, point fusarium Caulis et Folium Lini specialized form, phytophthora blight of pepper, tomato gray mould bacterium, Fusarium oxysporum, standing grain
Paddy Fusarium spp., fusarium moniliforme, Rhizoctonia cerealis, early epidemic pathogenic bacteria, gaeumannomyce bacterium or sclerotinite.
In a detailed description of the invention, the preparation of the fermentation liquid of described solution bacillus subtilis IBFCBF-4 includes: by withered
Grass bacillus cereus IBFCBF-4 is seeded in liquid fermentation medium, carries out fermentation culture at 25-30 DEG C, and shaking table shakes 2-4 days,
Obtain the fermentation liquid of bacillus subtilis IBFCBF-4.
In a detailed description of the invention, described liquid fermentation medium comprises following components according to concentration: 8～12g/L
Tryptone, the beef powder 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.
The bacillus subtilis strain IBFCBF-4 that the present invention provides has that antifungal scope is wide, antagonistic effect is the most special
Property, thus there is good industrial applications prospect.Meanwhile, the bacillus subtilis strain IBFCBF-4 that the present invention provides is to agriculture
Crop has good growth-promoting effect.
Especially, IBFCBF-4 bacterium former to Oil Tea Anthracnose has obvious inhibitory action, and field test shows IBFCBF-
The prevention effect of 4 pairs of oil tea anthraxs is significantly higher than IBFCBF-1.
Accompanying drawing explanation
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
In having technology to describe, the required accompanying drawing used is briefly described, it should be apparent that, those of ordinary skill in the art are come
Say, on the premise of not paying creative work, it is also possible to obtain other accompanying drawing according to these accompanying drawings.
Fig. 1 is the preparation flow figure of the bacillus subtilis IBFCBF-4 that the embodiment of the present invention provides；
Fig. 2 is the colonial morphology figure of the bacillus subtilis IBFCBF-4 that the embodiment of the present invention provides；
Fig. 3 is the Gram’s staining figure of the bacillus subtilis IBFCBF-4 that the embodiment of the present invention provides；
Fig. 4 is the spore staining figure of the bacillus subtilis IBFCBF-4 that the embodiment of the present invention provides；
Fig. 5 is the PCR primer sepharose electrophoresis figure of the bacillus subtilis IBFCBF-4 that the embodiment of the present invention provides；
Fig. 6 is the phylogenetic tree of the 16S sequence construct of the bacillus subtilis IBFCBF-4 that the embodiment of the present invention provides
Figure；
Fig. 7 is bacillus subtilis IBFCBF-4 and the IBFCBF-1 flat board antagonism oil tea anthrax of embodiment of the present invention offer
Pathogen contrast schematic diagram；
Fig. 8 is the schematic diagram of the bacillus subtilis IBFCBF-4 preventing and treating capsicum epidemic disease that the embodiment of the present invention provides.
Detailed description of the invention
The bacillus subtilis strain that the embodiment of the present invention provides, makes bacillus subtilis that the present invention provides and is given birth to
Plant silborne fungal diseases can effectively be prevented and treated by the fermentation liquid produced.
For the technical scheme making those skilled in the art be more fully understood that in the embodiment of the present invention, and make the present invention real
Execute the above-mentioned purpose of example, feature and advantage can become apparent from understandable, below in conjunction with the accompanying drawings to the technology in the embodiment of the present invention
Scheme is described in further detail.
The bacillus subtilis strain that the embodiment of the present invention provides is bacillus subtilis IBFCBF-4, described hay spore
Bacillus IBFCBF-4 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on August 11st, 2015
(it is called for short 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.
Refer to accompanying drawing 1, accompanying drawing 1 shows the preparation stream of the bacillus subtilis IBFCBF-4 that the embodiment of the present invention provides
Cheng Tu.The preparation process of the bacillus subtilis IBFCBF-4 that the embodiment of the present invention provides is:
S01: gather soil sample by multi-point sampling method, and by finely ground for the soil sample that collects, in the embodiment of the present invention soil sample
Take from Changling, salt-soda soil pH value 10.0, saline and alkaline 0.65%.；
S02: finely ground soil sample put into and fully shake equipped with in the centrifuge tube of sterilized water, making concentration is 10-1Soil
Suspension；
S03: by described Soil Slurry gradient dilution, obtain the Soil Slurry of variable concentrations；
S04: choosing concentration is 10-5、10-6With 10-7Soil Slurry, and join and be coated with on NA culture medium flat plate
Cloth processes, and is put in the incubator of 30 DEG C cultivation, obtains bacterium colony；
S05: select single bacterium colony that form differs and carry out conservation of ruling, cultivate 24 hours, obtains separation bacterium, by described separation
Bacterium is placed in the refrigerator of 4 DEG C and saves backup；
S06: with the card punch of diameter 0.5cm, Caulis et Folium Lini stood withered, Fructus Capsici epidemic disease enzyme, Oil Tea Anthracnose former bacterium truffle and inoculate respectively
In different PDA culture medium flat plate center (it is as a comparison that Caulis et Folium Lini founds withered, Fructus Capsici epidemic disease enzyme truffle), at anomaly plate central authorities 2.5cm
The separation bacterium that at decussation, equidistant inoculation is different, not inoculate separation bacterium for comparison (CK), processes every time and carries out 3 times and repeat
Experiment, cultivates under the conditions of temperature is 25 DEG C, when matched group (CK) covers with whole vessel, and the size of record inhibition zone, select
Bacterium inhibition zone former to Oil Tea Anthracnose maximum for bacillus subtilis IBFCBF-4.
Wherein, NA culture medium is nutrient agar, and the composition of this nutrient agar includes 10g/L according to concentration
Tryptone, the beef powder of 3g/L, the agar of the NaCl of 5g/L, 20g/L.NA culture medium pH value in the preparation be 7.2～
7.4, and through the sterilization treatment of 20min, and temperature during sterilizing is 121 DEG C.PDA culture medium is potato dextrose agar training
Support base.
Embodiments provide bacillus subtilis IBFCBF-4 and IBFCBF-1 (number of patent application
201510867702.4) the contrast schematic diagram of the former bacterium of antagonism Oil Tea Anthracnose, schematic diagram refer to accompanying drawing 7, and (left side is IBFCBF-
4).From accompanying drawing 7 it can be seen that when matched group all covers with vessel, it is inoculated with bacillus subtilis IBFCBF-4 and separates bacterium
PDA culture medium on occur in that obvious inhibition zone, and the diameter of inhibition zone is all at more than 2.0cm, and its inhibition zone size is notable
Higher than IBFCBF-1 inhibition zone size 1.2cm to oil tea anthrax.Carried by the explanation embodiment of the present invention that goes out to be now able to of inhibition zone
The bacillus subtilis IBFCBF-4 bacterium former to Oil Tea Anthracnose of confession has obvious inhibitory action, it is thus possible to well apply
In preventing and treating fungal disease, field test shows that IBFCBF-4 reaches 74.40% to the prevention effect of oil tea anthrax, is significantly higher than
The prevention effect 40.69% of IBFCBF-1.
After table 1:IBFCBF-1 Yu IBFCBF-4 process, a situation arises for Oil Tea Anthracnose
| Process | Disease index before dispenser | 14d disease index after dispenser | Prevention effect (%) |
| Clear water compares | 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 prevention effect %=[(disease index after disease index-matched group dispenser before matched group dispenser)-(process group
Disease index after disease index-process group dispenser before dispenser)]/(state of an illness after disease index-matched group dispenser before matched group dispenser
Index) × 100.
The preparation of the fermentation liquid of the bacillus subtilis IBFCBF-4 that the embodiment of the present invention provides includes: by bacillus subtilis
Bacterium IBFCBF-4 is seeded in liquid fermentation medium, carries out fermentation culture at 25-30 DEG C, and shaking table shakes 2-4 days, obtains hay
The fermentation liquid of bacillus cereus IBFCBF-4.
Wherein, the rotating speed of shaking table concussion is 180r/min.
Liquid fermentation medium comprises the tryptone of following components: 10g/L according to concentration, the beef powder of 3g/L, 20g/L
Glucose, the NaCl of 5g/L.Liquid fermentation medium pH value in the preparation is 7.2～7.4, and through the sterilizing of 20min
Process, and temperature during sterilizing is 121 DEG C.
The bacterial strain that the embodiment of the present invention is provided by the present invention is identified, this qualification includes herein below:
1, Morphological Identification
The bacterial strain that the embodiment of the present invention provides is scoring on NA culture medium flat plate, then flat board is reversed, in temperature be
Cultivate 24h under conditions of 30 DEG C, observe and record the growing state of bacterium colony on flat board.The bacterium of the bacterial strain that the embodiment of the present invention provides
The aspect graph that falls refer to accompanying drawing 2.
It can be seen that the bacterium colony of bacterial strain that the embodiment of the present invention provides is slightly yellow from accompanying drawing 2, opaque, circular or
Irregular shape, edge is irregular, rough surface fold.
Further, the bacterial strain provided the embodiment of the present invention with test kit carries out Gram’s staining and spore staining, and
Oil Microscopic observation bacterial strain and bacterial strain is taken pictures.The Gram’s staining of this bacterial strain and spore staining refer to accompanying drawing 3 and accompanying drawing
4。
From accompanying drawing 3 it can be seen that after Gram’s staining, the bacterial strain that the embodiment of the present invention provides is shaft-like, and in indigo plant
Purple, for gram positive bacteria.From accompanying drawing 4 it can be seen that after spore staining, the bacterial strain thalline that the embodiment of the present invention provides
Aobvious blueness, the aobvious redness of spore, the bacterial strain that thus the explanation present invention provides can produce spore.
2, Physiology and biochemistry is identified
(1) catalase experiment
In the liquid medium of bacterial strain, the hydrogen peroxide of directly dropping 3%, observes immediately.If there being a large amount of bubble to produce,
It is then positive；If not producing bubble, then it is negative.The bacterial strain that the present invention provides produces a large amount of bubble immediately, and experimental result is sun
Property.
(2) oxidase experiment
Extracting waste cleaning filter paper one jiao, dips a small amount of bacterial strain bacterium colony, and adding concentration is that the hydrochloride base of 1% is to benzene two
Amine aqueous solution one, positive pinkiness immediately, and color can gradually be deepened.In this is tested, bacterium colony pinkiness, face
Color is gradually deepened, and experimental result is positive.
(3) Starch Hydrolysis experiment
The strain point of bacterial strain is received on starch culture-medium, under conditions of temperature is 37 DEG C, cultivates 24h, drips a small amount of iodine
Liquid, on starch culture-medium flat board, rotates gently, makes iodine liquid be evenly distributed on starch culture-medium flat board, observes periphery of bacterial colonies
Situation.If periphery of bacterial colonies occurs that water white transparency circle shows there is amylatic ability, otherwise, then do not have.The week of this bacterial strain bacterium colony
It is with transparent circle to produce, it is possible to illustrate that this bacterial strain has amylatic ability.
(4) C.I. 13020. MR experiment
This bacterial strain that picking is a small amount of, and be inoculated on collective media, cultivate 3～5 days under conditions of temperature is 30 DEG C,
Cultivation takes culture fluid 1ml after terminating, and adds methyl red indicator 1～2, and positive in cerise, the weak positive is pale red, cloudy
Property is yellow.In this is tested, bacterium solution turns yellow, so being negative.
(5) VP experiment
This bacterial strain that picking is a small amount of, and it is inoculated in collective media, cultivate 4 days under conditions of temperature is 30 DEG C, cultivate
Take culture fluid 2.5ml after end and be initially charged a naphthols absolute alcohol solution 0.6ml, add the potassium hydroxide that concentration is 40% water-soluble
Liquid 0.2ml, shakes 2-5min, and positive bacteria presents redness the most immediately, if redfree occurs, is statically placed in room temperature or the constant temperature of 30 DEG C
In case, if the most not manifesting redness in 2h, then can determine that as feminine gender.In this is tested, bacterium solution reddens immediately, so being positive.
(6) gelatin liquefaction experiment
Taking this bacterial strain, percutaneous puncture-inoculation is in gelatin, and makes this bacterial strain be positioned at the 2/3 of the gelatin degree of depth.It it is 20 DEG C in temperature
Under conditions of cultivate 5-7d.Every day, whether this bacterial strain of observed result was liquefied by antibacterial, if being liquefied, then for the test positive；If no
It is liquefied, is then negative.In this is tested, gelatin is liquefied, and therefore this bacterial strain is positive.
(7) nitrate reduction experiment
By this inoculation in nitrate broth, under conditions of temperature is 28 DEG C, 3d cultivated by shaking table, then takes 5mL training
Nutrient solution, is illustrated to add developer by test kit (Hai Bo Bioisystech Co., Ltd nitrate reduction test kit), turns yellow as the positive,
Otherwise, do not change color as feminine gender.In this is tested, bacterium solution turns yellow, and illustrates that this bacterial strain is for the positive.
(8) hydrogen sulfide experiment is produced
By this bacterial strain percutaneous puncture-inoculation in lead acetate medium, under conditions of temperature is 35 DEG C, cultivates 24～48h, and sees
Examine result.If culture medium blackening, then it is positive；Not blackening, then be negative.In this is tested, culture medium variable color, this bacterium is described
Strain is positive.
(9) citrate utilizes experiment
Choose the streak inoculation on simon Si Shi citrate medium inclined-plane of this bacterial strain, train under conditions of temperature is 37 DEG C
Support 3-7 days.If culture medium to be alkalescence person, i.e. indicator blue or pinkish be positive；If culture medium invariant color, then it is cloudy
Property.In this is tested, the non-variable color of culture medium, illustrate that this bacterial strain is for feminine gender.
(10) lecithin activity experiment
The surface of Fresh Egg is carried out disinfection by the ethanol with 75%, and with the tweezers of sterilizing, egg is beaten a hole, inclines
Ovum Gallus domesticus album, then with aseptic straw sucking-off yolk, is cooled to after joining thawing in the NA culture medium of about 50 DEG C, after being mixed evenly
Fell flat board, and point connects this bacterial strain, cultivates 24h, and observe under conditions of temperature is 30 DEG C.If there is muddiness in colony edge
Circle person is enzyme positive.In this is tested, there is the most muddy circle in colony edge, illustrates that this bacterial strain is for the positive.
(11) malonate utilizes experiment
Picking is cultivated 12h lawn and is inoculated in malonate culture medium, cultivates 24-48h, training under conditions of temperature is 35 DEG C
Support base by green change indigo plant person into positive, otherwise be negative.In this is tested, culture medium variable color, illustrate that this bacterial strain is for the positive.
(12) glucose fermentation experiment
A small amount of this bacterial strain percutaneous puncture-inoculation of picking, in glucose oxidation-fermentation medium, is cultivated under conditions of temperature is 30 DEG C
3d, observes the change of culture medium color.If changing without color, then needing to continue to observe 7d, culture medium flavescence person is fermented type.?
This time in experiment, culture medium turns yellow, and illustrates that this bacterial strain is for the positive.
(13) cellulose decomposition experiment
Spread plate is on sodium carboxymethyl cellulose solid medium, in time having obvious bacterium colony, instills one in flat board
Pipe Congo red dye liquor, and make Congo red dye liquor be evenly distributed on flat board.Add 1ml sodium chloride solution after 15 minutes, soak
After 15 minutes, wash away dyeing, see whether to produce transparent circle.If there being transparent circle to produce, then it is positive.In this is tested, have
Transparent circle produces, and illustrates that this bacterial strain is for the positive.
(14) the galactose utilization experiment positive
By this inoculation in galactose culture medium, under conditions of temperature is 30 DEG C, cultivates 2d, observes colony growth
Situation, if bacterium colony is formed, then can utilize galactose, otherwise, not all right.In this is tested, thalli growth, illustrate that this bacterial strain can
To utilize galactose.
(15) arabinose utilizes experiment
By this inoculation in arabinose culture medium, under conditions of temperature is 30 DEG C, cultivates 2d, observes bacterium colony raw
Long situation, if bacterium colony is formed, then can utilize arabinose, otherwise, not all right.In this is tested, thalline does not grows, and this is described
Bacterial strain cannot utilize arabinose.
(16) mannose utilizes experiment
By this inoculation in mannose culture medium, under conditions of temperature is 30 DEG C, cultivates 2d, observes colony growth
Situation, if bacterium colony is formed, then can utilize mannose, otherwise, not all right.In this is tested, thalli growth, this bacterial strain can be in order to
Use mannose.
(17) D-Fructose utilizes experiment
By this inoculation in D-Fructose culture medium, under conditions of temperature is 30 DEG C, cultivates 2d, observes colony growth
Situation, if bacterium colony is formed, then can utilize D-Fructose, otherwise, not all right.In this is tested, thalli growth, this bacterial strain can be in order to
Use D-Fructose.
(18) D-xylose utilization experiment
By this inoculation in D-xylose media, under conditions of temperature is 30 DEG C, cultivates 2d, observes colony growth
Situation, if bacterium colony is formed, then can utilize D-xylose, otherwise, not all right.In this is tested, having bacterium colony to be formed, this bacterial strain is permissible
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 |
| Catalase measures | + | Citrate utilizes | + |
| Oxidase assay | + | Lecithin activity measures | + |
| Starch Hydrolysis measures | + | Malonate utilizes | + |
| C.I. 13020. MR measures | — | Cellulose decomposition | + |
| VP tests | + | Galactose utilization | + |
| Gelatin liquefaction measures | + | Arabinose utilizes | — |
| Nitrate reduction measures | + | Mannose utilizes | + |
| Glucose fermentation | + | D-Fructose utilizes | + |
| Product hydrogen sulfide measures | — | D-xylose utilization | + |
Wherein ,+be expressed as this bacterial strain and respond and maybe can utilize ,-be expressed as this bacterial strain and do not react and maybe cannot utilize.
3,16S rDNA sequence analysis
The embodiment of the present invention uses health to be that century bio tech ltd's genome extraction test kit extracts in this bacterial strain
DNA.
Wherein, said extracted test kit includes the PCR reaction system of 50 μ L, and PCR reaction system include 25 μ L 2 ×
Master Mix；The forward 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 is: be 94 DEG C of denaturations 5min in temperature；94 DEG C of degeneration 0.5min；53 DEG C of annealing 0.5min；72
DEG C extend 1min；30 circulations, 72 DEG C extend 5min, 4 DEG C of preservations.
Refer to accompanying drawing 5, accompanying drawing 5 shows that the PCR primer of the bacillus cereus IBFCBF-4 that the embodiment of the present invention provides is used
The electrophoretogram of 1% agarose gel electrophoresis detection.PCR primer is checked order by your bio tech ltd of dimension generation, Changsha, surveys
Sequence result sees the nucleotide sequence in subordinate list.Gained sequence is carried out homology sequence comparison analysis by NCBI BLAST,
Obtain the sequence that similarity is higher.Using MEGA6.06 software building phylogenetic tree, the 16S rDNA sequence of this bacterial strain is with withered
The homology of grass bacillus cereus (Bacillus subtilis) reaches 100%, and the growth tree graph of this bacterial strain refer to accompanying drawing 6.
Summary morphological observation, Physiology and biochemistry are identified and 16S rDNA the sequencing results, it is possible to determine this bacterial strain
IBFCBF-4 is bacillus subtilis Cordycepps bacillus cereus (Bacillus subtilis), and it is named as bacillus subtilis
IBFCBF-4。
Bacillus subtilis IBFCBF-4 and fermentation liquid thereof that the embodiment of the present invention provides can be applied to prevent and treat mycosis
Evil, it is Rhizoctonia solani Kuhn (Rhizoctonia solani), Caulis et Folium Lini anthrax that this fungal disease passes fungus diseases for soil
(Colletotrichum linicola), point fusarium Caulis et Folium Lini 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 pathogenic bacteria (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 fermentation liquid thereof to very as a example by phytophthora blight of pepper
The research of fungus diseases, research contents is as follows.
(1) selection of culture medium
The cultivation of bacillus subtilis IBFCBF-4 uses NB fluid medium (i.e. beef extract-peptone fluid medium),
The preparation of this NB fluid medium comprises the peptone of 10g, the beef powder of 3g and the NaCl of 5g, and above-mentioned substance uses deionized water
It is settled to 1000ml, and to regulate pH value be 7.2-7.4, then 1 × 105Sterilizing 20min under the pressure of Pa, prepares the training of NB liquid
Support base.
Fructus Capsici epidemic disease enzymophathy fungal pathogens uses PDA fluid medium, and the preparation of this PDA fluid medium includes the peeling of 200g
Rhizoma Solani tuber osi, the glucose of 20g, above-mentioned substance uses deionized water to be settled to 1000mL, then 1 × 105Go out under the pressure of Pa
Bacterium 20min, prepares PDA fluid medium.
(2) selection of capsicum variety
Fructus Capsici selects the Sweet Pepper Varieties eggplant door that Vegetable Research Inst., Hunan Prov. Agriculture Science Academy provides.By the seed of eggplant door with dense
Degree be 10% hydrogen peroxide carry out surface sterilization 20min, then by sterilized water shower 3 times, and be positioned overCulture dish
In, constant temperature half-light accelerating germination under conditions of temperature is 30 DEG C.The seed choosing a length of 0.5cm of bud is sowed in 9 hole alms bowls, and substrate is city
Selling Nutrition Soil, Fructus Capsici is placed in the phjytotron of 30 ± 1 DEG C and cultivates, and the intensity of now illumination is 12000Lux, illumination
Cycle is L D=14 10, RH=80% ± 10%.
(3) specific experiment design
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 rotating speed is cultivation 48h under conditions of 180r/min,
Then with sterilized water, bacterium solution is diluted to 105CFU/mL.Fructus Capsici elicitin pathogenic bacteria is 30 DEG C in temperature, and rotating speed is 180r/min's
Under the conditions of cultivate 7d, then with sterilized water, bacterium solution is diluted to 105CFU/mL。
When experiment, the pepper seed one sprouted is sowed in 9 Bo Zhongmei holes, hole, it is ensured that 9 alms bowl Hot Pepper Seedling survivals, and by 9 alms bowls
Hot Pepper Seedling is divided into three groups, often organizes three alms bowls, and when Fructus Capsici was cultivated to the 3-4 leaf phase, the rhizosphere in Fructus Capsici applies Fructus Capsici elicitin pathogenic bacteria
Bacterium solution 2.5mL, then to being divided into NB culture medium, clear water and the bacillus subtilis adding 2.5mL in the young plant of three groups respectively
IBFCBF-4 bacterium solution.Applying 5mL nutritional solution every the every hole of 2d, to ensure that substrate is the most moistening, Fructus Capsici can normal growth.Observe
Chili growth situation, carries out to Fructus Capsici after 60d that plant height, stem be thick, interval, chlorophyll content in leaf blades, top half fresh weight and dry
Weight and the mensuration of the growth indexes such as root system fresh weight and dry weight, statistics often group capsicum epidemic disease morbidity strain number.
(4) measuring method
During measurement, the distance between pepper plant base portion to stem top i.e. trunk diameter growth point is plant height, the nearly root knot of plant
A diameter of stem of first segment stem thick.Random three leaves of often selecting good strains in the field for seed, measure its chlorophyll content with chlorophyll meter.Plant is the least
The a length of interval of joint stem, first three joint of nearly root knot is chosen in this experiment.Pepper plant is cut from first node of nearly root knot
Disconnected, weigh top half and the weight of root system part respectively, be designated as fresh weight, be then placed in the baking oven of 85 DEG C drying to permanent
Weight, weighs its weight the most respectively, is designated as their dry weight.Experimental result refer to table 3, table 4 and accompanying drawing 8.
The impact on chili growth of 3: the three kinds of different disposal methods of table
Table 4: a situation arises for the capsicum epidemic disease processed with three kinds of distinct methods
| Process | Morbidity strain number (strain) | Sickness rate (%) | Prevention effect (%) |
| 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/investigate total strain number × 100；(the 1-process group morbidity strain of prevention effect %=
Rate/matched group diseased plant rate) × 100.
It can be seen that the process of T group makes Fructus Capsici have higher plant height, a thicker stem from table 2, and second section pitch,
3rd 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 want big.Meanwhile, it can be seen that pepper plant grows fine from accompanying drawing 8, thus, it is withered that the embodiment of the present invention provides
Grass bacillus cereus IBFCBF-4 and fermentation liquid thereof can be effectively promoted the growth of plant.From table 3 it can be seen that at T group
Reason makes the sickness rate of the Fructus Capsici sickness rate significantly lower than other two groups, and prevention effect is significantly greater than the preventing and treating of other two groups especially
Effect, it is possible to illustrate that the bacillus subtilis IBFCBF-4 of embodiment of the present invention offer and fermentation liquid thereof have the most short of money
Anti-effect.
Invention described above embodiment, is not intended that limiting the scope of the present invention.Any in the present invention
Spirit and principle within amendment, equivalent and the improvement etc. made, should be included within the scope of the present invention.
Claims (10)
1. a bacillus subtilis strain, it is characterised in that described bacillus subtilis strain is bacillus subtilis
IBFCBF-4, and described bacillus subtilis IBFCBF-4 is preserved in the common micro-life of China Committee for Culture Collection of Microorganisms
Thing center, deposit number is CGMCC No.11233.
Bacillus subtilis strain the most according to claim 1, it is characterised in that described bacillus subtilis IBFCBF-4
Bacterium colony be slightly yellow, opaque, circular or irregular shape, edge is irregular, rough surface fold.
3. the preparation method of the bacillus subtilis IBFCBF-4 described in a claim 1 or 2, it is characterised in that described system
Preparation Method comprises the following steps:
Step 1, gathers soil sample, and soil sample requires salt-soda soil pH value 9.5～10.5, saline and alkaline 0.60～0.70%；
Step 2, makes the Soil Slurry of variable concentrations by soil sample；
Step 3, joins described Soil Slurry and is coated on NA culture medium flat plate processing, cultivate and obtain bacterium colony；
Step 4, selects single bacterium colony that form differs and carries out conservation of ruling, and cultivates and obtains separating bacterium；
Step 5, is inoculated into the center of PDA culture medium flat plate by pathogen truffle, described away from the inoculation of plate center equidistant
Separate bacterium, select after cultivation inhibition zone maximum for bacillus subtilis IBFCBF-4.
The preparation method of bacillus subtilis IBFCBF-4 the most according to claim 3, it is characterised in that described pathogen
Truffle is Oil Tea Anthracnose former bacterium truffle.
The preparation method of bacillus subtilis IBFCBF-4 the most according to claim 3, it is characterised in that step 3 is cultivated
Temperature is 28～32 DEG C, and step 5 cultivation temperature is 23～27 DEG C.
6. a right wants the application of the bacillus subtilis IBFCBF-4 described in 1 or 2, it is characterised in that described hay spore
The fermentation liquid of bacillus IBFCBF-4 and described bacillus subtilis IBFCBF-4 is applied to prevent and treat fungal disease.
The application of bacillus subtilis IBFCBF-4 the most according to claim 6, it is characterised in that described fungal disease is
Soil passes fungus diseases.
The application of bacillus subtilis IBFCBF-4 the most according to claim 7, it is characterised in that described soil passes fungus diseases
For the former bacterium of Oil Tea Anthracnose, Rhizoctonia solani Kuhn, Caulis et Folium Lini anthrax, point fusarium Caulis et Folium Lini specialized form, phytophthora blight of pepper, tomato gray mould
Bacterium, Fusarium oxysporum, Fusarium graminearum, fusarium moniliforme, Rhizoctonia cerealis, early epidemic pathogenic bacteria, gaeumannomyce bacterium or sclerotinite.
The application of bacillus subtilis IBFCBF-4 the most according to claim 6, it is characterised in that described solution hay spore bar
The preparation of the fermentation liquid of bacterium IBFCBF-4 includes: be seeded in liquid fermentation medium by bacillus subtilis IBFCBF-4,
25-30 DEG C carries out fermentation culture, and shaking table shakes 2-4 days, obtains the fermentation liquid of bacillus subtilis IBFCBF-4.
The application of bacillus subtilis IBFCBF-4 the most according to claim 9, it is characterised in that described liquid fermentation
Culture medium comprises following components according to concentration: the tryptone of 8～12g/L, the beef powder of 2～4g/L, the Fructus Vitis viniferae of 15～25g/L
Sugar, the NaCl of 4～6g/L, the pH of liquid fermentation medium are 7.2-7.4.
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| CN113801804A (en) * | 2021-03-24 | 2021-12-17 | 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 |
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| CN110819565A (en) * | 2019-11-20 | 2020-02-21 | 青岛农业大学 | Salt-tolerant bacillus BW9 with plant pathogenic bacteria inhibiting effect and application thereof |
| CN112831443A (en) * | 2021-02-22 | 2021-05-25 | 青岛农业大学 | Bacillus subtilis, microbial inoculum and application thereof in preventing and treating plant diseases |
| CN113801804A (en) * | 2021-03-24 | 2021-12-17 | 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 |
| 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 |
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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 |