CN109576179A - A kind of Bei Laisi Bacillus strain and its application in false smut - Google Patents
A kind of Bei Laisi Bacillus strain and its application in false smut Download PDFInfo
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Abstract
The present invention relates to microorganisms technical fields, and in particular to a kind of Bei Laisi Bacillus strain (Bacillus velezensis) QYQN-3, deposit number are as follows: 16567；And application of the bacterial strain in false smut.Bei Laisi bacillus of the invention has preferable control efficiency to rice green smut, is tested by 3 opposite culture methods, the results showed that Bei Laisi bacillus (Bacillus velezensis) QYQN-3 is 33.33mm to the inhibition zone of false smut.
Description
Technical field
The present invention relates to rice green smut Prevention Technique field, more particularly to a kind of Bei Laisi Bacillus strain and its
Application in false smut.
Background technique
False smut (Ustilaginoidea virens) is after the big Major Diseases rice blast of rice three, banded sclerotial blight and striped
The another main disease for influencing Rice Production after leaf blight.Raising, the cultivation of the quickening, Fertilization Level that are updated with rice varieties
The change of system, false smut gradually aggravate, and 20%~30% production loss can be caused when serious, have to rice yield very big
It influences.Toxin caused by ustilaginoidea virens generates serious hidden to people and animals' edible paddy safety and to the ecological safety of farm environment
Suffer from, so false smut is paid more and more attention.The history of false smut occurrence injury is short, research starting is late, especially in false smut
Prevention and treatment aspect, is reported very few.But it is largely very severe using influence of the chemical synthetic pesticide to environment, therefore rice is bent on rice
The biological control of disease is particularly important.Currently, had not been reported researcher filter out suitable bacterial strain come to false smut on rice into
Row biological control.
Summary of the invention
It is an object of the present invention to overcome the above-mentioned technical problems, and to provide a kind of Bei Laisi Bacillus strain and its in rice
Application in bent disease, can be fully solved above-mentioned technical problem.
The technical solution for solving above-mentioned technical problem is as follows:
A kind of Bei Laisi Bacillus strain (Bacillus velezensis) QYQN-3, deposit number are as follows: CGMCC
No.16567。
Furthermore it is also an object that a kind of screening technique of Bei Laisi Bacillus strain is provided, including with
Lower step:
Soil sample is taken in the paddy field of the rice main producing region of various regions, microorganism present in soil is isolated from soil sample
Bacterial strain carries out face-off experiment using 3 opposite culture methods and ustilaginoidea virens bacterial strain；
Bacterial strain QYQN-3 is filtered out, antagonistic effect is preferable；
Entitled Bei Laisi bacillus (the Bacillus velezen of the bacterium is indicated through morphological feature and molecular biology identification
sis)。
It further says, the specific steps of 3 opposite culture methods are as follows:
(1) culture medium of suitable growth of pathogenic bacteria is poured on culture dish；
(2) the ustilaginoidea virens bacteria cake that a diameter is 5mm is put in the centre of culture medium；
(3) bacteria cake that three strain to be tested QYQN-3 are put in the position of the central bacteria cake same distance of distance, makes three bacteria cakes
In a triangular arranged；
(4) diameter of inhibition zone is measured.
The application of the Bei Laisi Bacillus strain is to carry out biology to false smut using the Bei Laisi bacillus to prevent
It controls, passes through 3 opposite culture methods, suppression of Bei Laisi bacillus (Bacillus velezensis) QYQN-3 to false smut
Bacterium circle is 33.33mm.The meaning of inhibition zone is, indicates biocontrol microorganisms to the inhibiting effect of ustilaginoidea virens, the inhibition zone the big, says
The fungi-proofing inhibition to ustilaginoidea virens of open-birth is stronger, and control efficiency is better.
Above-mentioned strain has the feature that
1, morphological feature: bacterium colony is white or faint yellow, opaque, coarse to have fold, and cell is in direct rod shape.
2, physiological and biochemical property is as shown in the table:
In order to screen the efficient biocontrol microorganisms of ustilaginoidea virens, the present invention uses 3 opposite culture methods, to Liaoning Province, Sichuan Province
The paddy field soil sample acquired with areas such as the Inner Mongol is separated and is screened, and carries out Identification of Species to biocontrol bacterial strain.It is intended to obtain
The higher biocontrol bacterial strain of control efficiency is obtained, provides foundation for false smut biological control.
Compared with prior art, Bei Laisi bacillus of the invention has preferable control efficiency to rice green smut, leads to
Cross 3 opposite culture method tests, the results showed that Bei Laisi bacillus (Bacillus velezensis) QYQN-3 is to rice song
The inhibition zone of disease is 33.33mm.
Bei Laisi bacillus biological agent of the invention, will not pollute environment and ecology, be conducive to water
The No-harmful apple orchard of rice.
Detailed description of the invention
The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.
Fig. 1 is the big logotype of bacterium colony after false smut germ grows 27 days on PSA culture medium；
Fig. 2 is to be vaccinated with Bei Laisi bacillus (Bacillus velezensis) QYQN-3 around ustilaginoidea virens,
The big logotype of bacterium colony after grown 27 days on PSA culture medium；
Fig. 3 is the aspect graph of Bei Laisi bacillus (Bacillus velezensis) QYQN-3；
Specific embodiment
Embodiment 1:
A kind of Bei Laisi Bacillus strain (Bacillus velezensis) QYQN-3, deposit number are as follows: CGMCC
No.16567.Preservation date are as follows: on October 10th, 2018.Depositary institution are as follows: Institute of Microorganism, Academia Sinica.Preservation address
Are as follows: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
The screening technique of the Bei Laisi Bacillus strain, comprising the following steps:
Soil sample is taken in the paddy field of the rice main producing region of various regions, microorganism present in soil is isolated from soil sample
Bacterial strain carries out face-off experiment using 3 opposite culture methods and ustilaginoidea virens bacterial strain；
(1) culture medium of suitable growth of pathogenic bacteria is poured on culture dish；
(2) the ustilaginoidea virens bacteria cake that a diameter is 5mm is put in the centre of culture medium；
(3) bacteria cake that three strain to be tested QYQN-3 are put in the position of the central bacteria cake same distance of distance, makes three bacteria cakes
In a triangular arranged；
(4) diameter of inhibition zone is measured.
Bacterial strain QYQN-3 is filtered out, antagonistic effect is preferable；
Entitled Bei Laisi bacillus (the Bacillus velezen of the bacterium is indicated through morphological feature and molecular biology identification
sis)。
The Bei Laisi Bacillus strain has the feature that
A) morphological feature: bacterium colony is white or faint yellow, opaque, coarse to have fold, and cell is in direct rod shape (such as Fig. 3
It is shown)；
B) physiological and biochemical property is as shown in the table:
The detection method of the above physiological and biochemical property is as follows:
Gram's staining
The preparation of stain
A. liquid A and liquid B the mixed liquor of crystal violet: is prepared respectively, in which: solution A is crystal violet 2.0g, 95% ethyl alcohol
20ml；Second liquid is ammonium oxalate 0.8g, distilled water 80ml；Solution A second liquid is mixed, is filtered after standing 48 hours.This dye liquor is more steady
It is fixed, the several months can be stored in closed brown bottle.
B. iodine solution: first dissolving potassium iodide 2.0g with 3~5ml distilled water, then put into crystalline flake of iodine 1.0g, after iodine is completely dissolved,
Distilled water is added to be diluted to 300ml.
C. destainer: (1) 95% ethyl alcohol, (2) acetone ethanol solution: 95% ethyl alcohol 70ml and acetone 30ml.
D. redye liquid: using 2.5% safranin O ethanol solution as mother liquor, it is dilute that distilled water is added again with 1:4 volume ratio in when use
It releases.
Staining procedure
(1) a little lawn of transfer needle picking is used, is coated in a drop sterile water or the distilled water on clean slide, air-dries solid
It is fixed.
(2) it with after the mixed liquor dye 1min of crystal violet, is washed with water.
(3) iodine solution acts on 1min, and washing is blotted.
(4) it is decolourized with 95% ethyl alcohol or acetone ethanol solution, stream drops to eluent to colourless (about 30s).
(5) with liquid dye 2-3min is redyed, washing is air-dried.
Darkviolet is gram-positive bacterium；Red is gram-negative bacterium.
Sugar, alcoholic fermentation reaction
Culture medium: gemma bacterium culture medium (NH4)2HPO41.00g, KCl 0.20g, MgSO40.20g；Yeast extract 0.20g,
Agar 15.00g, sugar or alcohol 10.00g, 0.04% bromocresol purple 15.00ml, distilled water 1L, adjust pH 7.0~7.2；
0.04% bromocresol purple solution is that 0.04g bromocresol purple powder is taken to be dissolved in the dehydrated alcohol of 20ml, adds 80ml distillation
Water is formulated.With 18~young age culture percutaneous puncture-inoculation for 24 hours in above-mentioned culture medium, 28 DEG C of culture 7d.As indicator becomes
Huang indicates to produce acid, for the positive；Constant or change blue (purple) is then into feminine gender.
Motility
Semi-solid agar puncture method
1. culture medium
The phenomenon that can moving about in semisolid culturemedium according to bacterium amphitrichous but be unable to drift, observation are thin
Bacterium growing state, determines whether test organisms has motility.The culture medium that can be well grown using test organisms, wherein plus 0.4%
Agar, general semisolid culturemedium should be that the test tube that fell does not flow, and agar ruptures and is advisable when gently beaing on hand.
2. inoculation and observation
With staight needle percutaneous puncture-inoculation test organisms in semisolid culturemedium, thermophilic culture.The motility of bacterium can use transmitted light
Range estimation.If growth-gen is only grown in puncture line, edge is very clear, then it represents that test organisms without motion, for feminine gender；Such as growth
Object is in cloud diffusion by puncture line around, and edge is in cloud, then it represents that test strain has motility, for the positive.
Starch Hydrolysis reaction
Culture medium: add 0.2% soluble starch in NA culture medium；Lu's Ge Shi iodine solution: iodine 1.0g, potassium iodide 2.0g steam
Distilled water 300ml；Take fresh slant culture dibbling in above-mentioned plate, thermophilic culture.Culture 2-5 days, after forming obvious bacterium colony,
It is in blue edge color that iodine solution plate is added dropwise on plate, and periphery of bacterial colonies indicates that Starch Hydrolysis is positive if any non-discolouring transparent circle；It is still blue
Black is feminine gender.
Salt tolerance reaction
Selection is suitable for the fluid nutrient medium of bacterium to be measured growth, according to identification need to be added various concentration NaCl (3%, 5%,
7%, 10%, 20%), culture medium will be clarified extremely.With 18~young age culture for 24 hours is inoculated in above-mentioned culture medium, 28 DEG C
Cultivate 7d.It is compared with nonvaccinated control tube, observes growing state.It is grown to the positive, is not grown to feminine gender.
Contact enzyme reaction
The slant strains that will be cultivated for 24 hours take a small ring to be applied to the glass for having dripped and having had 10% hydrogen peroxide with platinum filament oese
On piece is generated if any bubble then for the positive, and bubble-free is feminine gender.
Cellulose hydrolysis
Culture medium: peptone water basal medium: peptone 5.00g, NaCl5.00g, distilled water 1L, pH 7.0~7.2；It will
Basal medium dispenses test tube, impregnates a high-quality filter paper in the medium.Paper slip width is advisable with fitting easily into test tube.Paper slip
Length about 5-7cm.When measuring aerobic bacteria, there should be part paper slip in outside culture medium liquid level, when measuring anaerobic bacteria, paper slip answers complete immersion
In culture medium.Inoculation medium should have the blank control not being inoculated with.It observes within thermophilic culture 1-4 weeks.Filter paper item can be resolved into
One fiber or by filter paper item fracture or thinning person for the positive, the unchanged person of filter paper item be feminine gender.
V-P measurement reaction
Culture medium: peptone 5g, glucose 5g, NaCl 5g, distilled water 1L, pH 7.0~7.2；Reagent: creatine 0.3%
Or original powder, NaOH40%；Inoculation test bacterium is in above-mentioned culture solution, and thermophilic culture 2,6 days；Take culture solution and 40% hydroxide
Sodium equivalent is mutually mixed.Add a little creatine, red, as test positive reaction, it is sometimes desirable to place longer occurs in 10min such as culture solution
Just there is red reaction in time.Do not occur red being then feminine gender.
Indoles
Culture medium: 1% tryptone aqueous solution；Adjust pH7.2~7.6；Fresh strain is inoculated in above-mentioned culture medium, Yu Shi
Temperature culture.Reagent: Paradimethylaminobenzaldehyde 8g, ethyl alcohol (95%) 760ml, dense HCl 160ml；Culture 1,2,4,7 days
Culture solution, at liquid layer interface red occurs for the reagent for being slowly added into 3-5mm high along tube wall in culture solution surface, as positive anti-
It answers.If color is unobvious, 4-5 drop ether can be added to culture solution, shaken, be scattered in ether in liquid, culture solution is stood into piece
It carves, adds indole reagent again after ether is floating to liquid level.When as having indoles in culture solution, indoles can be extracted in ether layer, dense
The indoles and reagent of contracting react, then color is obvious.
Nitrate reduction
Culture medium: liquid NA culture medium 1L, KNO31g；Reagent: 1) Ge Lisishi reagent: A liquid, p-aminobenzene sulfonic acid
0.5g, 10% spirit of vinegar 150ml；B liquid, alpha-naphthylamine 0.1g, distilled water 20ml, 10% spirit of vinegar 150ml；2) diphenylamines
Reagent: diphenylamines 0.5g is dissolved in the 100ml concentrated sulfuric acid, is diluted with 20ml distilled water.
Measurement bacterium is inoculated in nitrate fluid nutrient medium, sets thermophilic culture 1,3,5 days.Every plant of bacterium makees two repetitions,
Separately two pipes is stayed not to be inoculated with to compare.
It takes two clean empty test tubes or people cultivates 1,3,5 days culture solutions a little in colorimetric porcelain dish alveole, then respectively
Add a drop A liquid and B liquid, A liquid, each drop of B liquid are equally added in control tube.
When instilling A in culture solution, after B liquid, solution such as becomes pink, attacks the expression nitrous acid such as rare red, orange, brown
Salt exists, and is that nitrate reduction is positive.If redfree occurs, then it can add one, two drop diphenylamines reagents, at this time as blue anti-
It answers, then it represents that still have nitrate in culture solution, and reacted without nitrite, indicate nitrate-free reduction；If not blue
Reaction indicates that the nitrite of nitrate and formation has all been reduced into other substances, therefore should handle by the nitrate reduction positive.
Citrate
Culture medium: NaCl1g, MgSO4·7H2O0.2g, NH4H2PO40.5g, sodium citrate 2g, distilled water 1L, 0.04% phenol
Red liquid 20ml, the streak inoculation on inclined-plane, thermophilic culture 3-7 days.Culture medium is that alkalinity (indicator blue or pink) person is
Otherwise the positive is feminine gender.
Hydrogen sulfide generates
Paper strip method
Culture medium: peptone 10g, NaCl5g, beef extract 10g, cysteine 0.5g, distilled water 1L, pH7.0~7.4 point
Fill test tube, every pipe height of liquid layer 4-5cm, 112 DEG C of 20~30min of sterilizing.
In addition ordinary filter paper is cut into the paper slip of 0.5-1cm wide, length is depending on test tube and culture medium height.With 10%
Lead acetate paper slip is impregnated with, then dried with baking oven, be put in culture dish sterilize it is spare.
With fresh slant culture inoculation medium.After inoculation, a lead acetate paper slip tampon plug is taken with sterile sub-folder
Tightly, make to hang in pipe, lower end does not contact liquid level, thermophilic culture close to media surface.It observes within 3,7,14 days after inoculation.
The paper slip person of turning black is the positive, and constant person is feminine gender.
As seen from the above table, Gram's staining, glucose, sucrose, lactose, maltose, fructose, xylose, mannose, inositol,
Sorbierite, mannitol, motility, Starch Hydrolysis, salt tolerance 3%, salt tolerance 5%, salt tolerance 7%, salt tolerance 10%, indoles,
Citrate, nitrate reduction, V-P test are positive, and salt tolerance 20%, catalase, cellulose hydrolysis, hydrogen sulfide are generated in yin
Property.
Biological control is carried out to false smut using the Bei Laisi bacillus, passes through 3 opposite culture methods, Bei Laisi bud
Spore bacillus (Bacillus velezensis) QYQN-3 is 33.33mm to the inhibition zone of false smut.
The above is only presently preferred embodiments of the present invention, not does limitation in any form to the present invention, it is all according to
According to any simple modification to the above embodiments in technical spirit of the invention, equivalent variations, guarantor of the invention is each fallen within
Within the scope of shield.
Claims (5)
1. a kind of Bei Laisi Bacillus strain, which is characterized in that the Bei Laisi Bacillus strain is Bei Laisi gemma bar
Bacterium (Bacillus velezensis) QYQN-3, deposit number are as follows: 16567.
2. Bei Laisi Bacillus strain according to claim 1, which is characterized in that Bei Laisi Bacillus strain tool
It has the following characteristics that
A) morphological feature: bacterium colony is white or faint yellow, opaque, coarse to have fold, and cell is in direct rod shape；
B) physiological and biochemical property is as shown in the table:
。
3. a kind of screening technique of Bei Laisi Bacillus strain as described in claim 1, which is characterized in that including following step
It is rapid:
Soil sample is taken in the paddy field of the rice main producing region of various regions, microbial bacteria present in soil is isolated from soil sample
Strain carries out face-off experiment using 3 opposite culture methods and ustilaginoidea virens bacterial strain；
Bacterial strain QYQN-3 is filtered out, antagonistic effect is preferable；
The entitled Bei Laisi bacillus of the bacterium (Bacillus velezensis) is indicated through morphological feature and molecular biology identification.
4. the screening technique of Bei Laisi Bacillus strain according to claim 3, which is characterized in that described 3 points pairs
It stands erect the specific steps of cultivation are as follows:
(1) culture medium of suitable growth of pathogenic bacteria is poured on culture dish；
(2) the ustilaginoidea virens bacteria cake that a diameter is 5mm is put in the centre of culture medium；
(3) bacteria cake that three strain to be tested QYQN-3 are put in the position of the central bacteria cake same distance of distance, makes three bacteria cakes in one
A triangular arranged；
(4) diameter of inhibition zone is measured.
5. a kind of application of Bei Laisi Bacillus strain as described in claim 1, which is characterized in that utilize the Bei Laisi bud
Spore bacillus carries out biological control to false smut, passes through 3 opposite culture methods, Bei Laisi bacillus (Bacillus
Velezensis) QYQN-3 is 33.33mm to the inhibition zone of false smut.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111304136A (en) * | 2020-04-15 | 2020-06-19 | 江苏省家禽科学研究所 | Lead-resistant Bacillus belgii and separation method and application thereof |
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| KR20110119386A (en) * | 2010-04-27 | 2011-11-02 | 주식회사 진상 | Gene coding for cellulase from bacillus velezensis a-68 and production method of cellulase by transformed escherichia coli a-68 thereof |
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| CN111304136A (en) * | 2020-04-15 | 2020-06-19 | 江苏省家禽科学研究所 | Lead-resistant Bacillus belgii and separation method and application thereof |
| CN111304136B (en) * | 2020-04-15 | 2021-10-01 | 江苏省家禽科学研究所 | Lead-resistant Bacillus belgii and separation method and application thereof |
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