CN109439593A - A kind of bacillus pumilus preventing and treating false smut - Google Patents
A kind of bacillus pumilus preventing and treating false smut Download PDFInfo
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- CN109439593A CN109439593A CN201811519107.1A CN201811519107A CN109439593A CN 109439593 A CN109439593 A CN 109439593A CN 201811519107 A CN201811519107 A CN 201811519107A CN 109439593 A CN109439593 A CN 109439593A
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- bacillus pumilus
- false smut
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- 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
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- 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
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- 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
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- 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
Abstract
The present invention relates to rice green smut Prevention Technique fields, more particularly to a kind of bacillus pumilus for preventing and treating false smut, (Bacillus pumilus) ZHQN-6, deposit number are as follows: 16565.Bacillus pumilus of the invention has preferable control efficiency to rice green smut, is tested by 3 opposite culture methods, the results showed that bacillus pumilus (Bacillus pumilus) ZHQN-6 is 31.67mm to the inhibition zone of false smut.
Description
Technical field
The present invention relates to rice green smut Prevention Technique fields, more particularly to a kind of short and small gemma for preventing and treating false smut
Bacillus.
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.The raising of the quickening, Fertilization Level that update with rice varieties is planted
The change of training system, false smut gradually aggravate, and 20%~30% production loss can be caused when serious, is had to rice yield
Very big influence.Toxin caused by ustilaginoidea virens generates tight to people and animals' edible paddy safety and to the ecological safety of farm environment
Weight hidden danger, so false smut is paid more and more attention.The history of false smut occurrence injury is short, research starting is late, especially in rice song
In terms of the prevention and treatment of disease, report very few.But it is largely very severe using influence of the chemical synthetic pesticide to environment, therefore rice
The biological control of upper false smut is particularly important.Come currently, having had not been reported researcher and having filtered out suitable bacterial strain to rice on rice
Bent disease carries out biological control.
Summary of the invention
It is an object of the present invention to overcome the above-mentioned technical problems, and to provide it is a kind of prevent and treat false smut bacillus pumilus,
Above-mentioned technical problem can be fully solved.
The technical solution for solving above-mentioned technical problem is as follows:
A kind of bacillus pumilus preventing and treating false smut is easy to cultivate and control efficiency is preferable；The bacillus pumilus
For bacillus pumilus (Bacillus pumilus) ZHQN-6, deposit number are as follows: 16565.
Furthermore it is also an object that providing a kind of screening side of bacillus pumilus for preventing and treating false smut
Method, it is simple and easy, easy to operate, specifically includes the following steps:
Soil sample is taken in the paddy field of the rice main producing region of various regions, micro- life present in soil is isolated from soil sample
Object bacterial strain carries out face-off experiment using 3 opposite culture methods and ustilaginoidea virens bacterial strain；
Bacterial strain ZHQN-6 is filtered out, antagonistic effect is preferable；
The entitled bacillus pumilus of the bacterium (Bacillus pumilus) is indicated through morphological feature and molecular biology identification.
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 ZHQN-6 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 bacillus pumilus is to carry out biological control to false smut using the bacillus pumilus, passes through three
Point opposite culture method, bacillus pumilus (Bacillus pumilus) ZHQN-6 are 31.67mm to the inhibition zone of false smut,
Biocontrol effect is preferable.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 creamy white, and round, neat in edge is regular, surface wettability, smooth, bacterium colony convex
It grows out；Its cell is in rod-short.
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
It saves and the paddy field soil sample of the area acquisitions such as the Inner Mongol is separated and screened, and Identification of Species is carried out to biocontrol bacterial strain.It is intended to
The higher biocontrol bacterial strain of control efficiency is obtained, provides foundation for false smut biological control.
Compared with prior art, bacillus pumilus of the invention has preferable control efficiency to rice green smut, passes through
3 opposite culture method tests, the results showed that bacillus pumilus (Bacilluspumilus) ZHQN-6 is to the antibacterial of false smut
Circle is 31.67mm.
Strain of i (bacillus) pumilus category biological agent of the invention, will not pollute environment and ecology, be conducive to
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 bacillus pumilus ZHQN-6 around ustilaginoidea virens, after grown 27 days on PSA culture medium
The big logotype of bacterium colony；
Fig. 3 is the aspect graph of bacillus pumilus (Bacillus pumilus) ZHQN-6；
Specific embodiment
Embodiment 1:
A kind of bacillus pumilus preventing and treating false smut, bacillus pumilus are bacillus pumilus (Bacillus
Pumilus) ZHQN-6, deposit number are as follows: 16565.Preservation date are as follows: on October 10th, 2018, depositary institution: the Chinese Academy of Sciences
Institute of microbiology.Preservation address: the Institute of Microorganism, Academia Sinica of Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
The screening technique of the bacillus pumilus, comprising the following steps:
Soil sample is taken in the paddy field of the rice main producing region of various regions, micro- life present in soil is isolated from soil sample
Object 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 ZHQN-6 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 ZHQN-6 is filtered out, antagonistic effect is preferable；
The entitled bacillus pumilus of the bacterium (Bacillus pumilus) is indicated through morphological feature and molecular biology identification.
The strain has the feature that
A) morphological feature: bacterium colony is creamy white, and round, neat in edge is regular, surface wettability, smooth, bacterium colony convex
It grows out；Its cell is in rod-short (as shown in Figure 3).
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, distilled water is added again with 1:4 volume ratio in when use
Dilution.
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
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
Bacterial growth situation, 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 when gently beaing on hand
It is advisable.
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 life
Surplus 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
Black-and-blue is negative.
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
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 should be complete
It is soaked in culture medium.Inoculation medium should have the blank control not being inoculated with.It observes within thermophilic culture 1-4 weeks.It can be by filter paper item
Resolve into a 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 more occurs in 10min such as culture solution
Just occurs red reaction for a long 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, in
Thermophilic culture.Reagent: Paradimethylaminobenzaldehyde 8g, ethyl alcohol (95%) 760ml, dense HCl 160ml；Culture 1,2,4,7 days
Culture solution, be slowly added into the reagent of 3-5mm high in culture solution surface along tube wall, occur at liquid layer interface it is red, it is as positive
Reaction.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
For a moment, add indole reagent again after ether is floating to liquid level.When as having indoles in culture solution, indoles can be extracted in ether layer,
The indoles and reagent of concentration 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 such as rare red, orange, brown
Hydrochlorate 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
Reaction, then it represents that still have nitrate in culture solution, and reacted without nitrite, indicate nitrate-free reduction；If not in indigo plant
Colour response indicates that the nitrite of nitrate and formation has all been reduced into other substances, thus should be by the nitrate reduction positive at
Reason.
Methyl red test
Culture medium: peptone 5g, glucose 5g, NaCl5g, distilled water 1L, pH 7.0~7.2；Reagent: methyl red
0.1g, 95% ethyl alcohol 300ml, distilled water 200ml；Inoculation test bacterium is in above-mentioned culture solution, and thermophilic culture 2,6 days；It is cultivating
A drop methyl red reagent is added in liquid, red is methyl red test positive reaction, and yellow is negative reaction.
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 alkalinity (indicator blue or pink) person
It is otherwise feminine gender for the positive.
Hydrogen sulfide generates
Paper strip method
Culture medium: peptone 10g, NaCl5g, beef extract 10g, cysteine 0.5g, 1 L of distilled water, 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
Paper slip is impregnated with by 10% lead acetate, is 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, salt tolerance 3%, salt tolerance 5%, salt tolerance
7%, fructose, xylose, mannose, mannitol, motility, indoles, methyl red test, citrate, catalase, V-P, which are tested, is in
The positive, salt tolerance 10%, salt tolerance 20%, maltose, cellulose hydrolysis, inositol, sorbierite, nitrate reduction, starch water
Solution, hydrogen sulfide generation are negative.
Biological control, by 3 opposite culture methods, short and small gemma bar are carried out to false smut using the bacillus pumilus
Bacterium (Bacillus pumilus) ZHQN-6 is 31.67mm 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, all
Upper any simple modification to the above embodiments, equivalent variations according to the technical essence of the invention, each fall within of the invention
Within protection scope.
Claims (5)
1. a kind of bacillus pumilus for preventing and treating false smut, which is characterized in that the bacillus pumilus is bacillus pumilus
(Bacillus pumilus) ZHQN-6, deposit number are as follows: 16565.
2. the bacillus pumilus of prevention and treatment false smut according to claim 1, which is characterized in that the strain has following special
Sign:
A) morphological feature: bacterium colony is creamy white, and round, neat in edge is regular, surface wettability, smooth, the birth of bacterium colony convex
It is long；Its cell is in rod-short；
B) physiological and biochemical property is as shown in the table:
。
3. a kind of screening technique of the bacillus pumilus of prevention and treatment false smut as described in claim 1, which is characterized in that including
Following steps:
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 ZHQN-6 is filtered out, antagonistic effect is preferable；
The entitled bacillus pumilus of the bacterium (Bacillus pumilus) is indicated through morphological feature and molecular biology identification.
4. the screening technique of the bacillus pumilus of prevention and treatment false smut according to claim 3, which is characterized in that described
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 ZHQN-6 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 bacillus pumilus as described in claim 1, which is characterized in that utilize the bacillus pumilus pair
False smut carries out biological control, passes through 3 opposite culture methods, ZHQN-6 pairs of bacillus pumilus (Bacillus pumilus)
The inhibition zone of false smut is 31.67mm.
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