CN108504603A - The biocontrol bacterial strain and screening technique of a kind of inducing paddy rice blast resisting and application - Google Patents
The biocontrol bacterial strain and screening technique of a kind of inducing paddy rice blast resisting and application Download PDFInfo
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Abstract
The invention discloses provide a kind of screening technique of the biocontrol bacterial strain of inducing paddy rice blast resisting, by the rice terrace soil sample for choosing the rice main producing region that the multiple rice blast in Liaoning Province are fallen ill different, pass through soil dilution planting, seed pelleting processing and the experiment of live leaves method, the results showed that bacillus megaterium reaches 71.97% to the control effect of inducing paddy rice blast resisting.Compared with prior art, biocontrol bacterial strain category biological agent of the invention, will not pollute environment and ecology, be conducive to the No-harmful apple orchard of rice.
Description
Technical field
The present invention relates to rice blast Prevention Technique field, especially a kind of biocontrol bacterial strain of inducing paddy rice blast resisting
And screening technique and application.
Background technology
Rice blast (Pyricularia oryzae) is a kind of sudden strong, is easy to popular important disease, main in the whole world
There is generation in the country of production rice.People use anti-pest kind and chemical prevention as main control measure at present, due to anti-
The resistance of pest kind, which can gradually weaken, even to be lost, and the residual of chemical agent can destroy ecological environment and the health of people and animals, and
In the governance process of plant disease induce plant generate disease resistance biocontrol microorganisms have control effect significantly, ecological environment security
Property height and many advantages, such as pathogen will not be induced to develop immunity to drugs, so in the prevention and control field of plant disease, induction plant production
The biocontrol bacterial strain of raw disease resistance has greatly research and development potentiality.
Invention content
The invention aims to solve the deficiencies in the prior art, a kind of life of inducing paddy rice blast resisting is provided
Anti- bacterial strain and screening technique and application.
In order to achieve the above objectives, the present invention is implemented according to following technical scheme：
A kind of biocontrol bacterial strain of inducing paddy rice blast resisting, the biocontrol bacterial strain are bacillus megaterium (Bacillus
Megaterium) DdN5-6, preserving number are：CGMCCNo.15457.
Further technical solution is that the culture medium of the biocontrol bacterial strain is NA culture mediums, the growth of biocontrol bacterial strain mycelia
Preference temperature is 28 DEG C.
Furthermore it is also an object that providing a kind of screening side of the biocontrol bacterial strain of inducing paddy rice blast resisting
Method includes the following steps：
Edaphon separation and indicator strain are carried out using soil dilution planting：Choose the multiple rice blast hairs in Liaoning Province
The rice terrace soil sample of sick different rice main producing region, successively dilution prepare 10-3、10-4、10-5、10-6Soil dilution liquid, by soil
Dilution takes 0.5mL to be applied to respectively in NA culture medium flat plates respectively, and each 3 ware of concentration culture is placed in 28 DEG C of insulating boxs and trains
It supports and obtains magnaporthe grisea spore suspension；After cultivating 3~4d on NA culture medium flat plates, to the bacterium grown on NA culture medium flat plates
Capable separation is dropped into, the different single bacterium colony scribed line culture of the cultural colonies such as picking form, color, purifying are then transferred to NA trainings
It supports to be put into 4 DEG C of refrigerators after being further cultured for 5d in base inclined-plane and save backup；Instruction biocontrol bacterial strain is bacillus megaterium (Bacillus
megaterium)。
Secondly, it is a further object of the invention to provide a kind of biocontrol bacterial strains of inducing paddy rice blast resisting in induction water
Application in terms of the prevention of rice blast resisting, specifically includes：
1) seed treatment：The biocontrol bacterial strain that separation obtains is transferred on NA culture medium flat plates and activates 72h at 28 DEG C, so
After be put into NB culture mediums, at 28 DEG C, 180rmin-1Shaker fermentation 3d, obtain isolated strains zymotic fluid, by 1:20 ratio
Example is to rice paddy seed difference Cotton seeds 2d；
2) live body Seedling Inoculation：It takes the rice paddy seed after aforementioned processing to carry out indoor pot, waits for that rice seedlings are grown to 3 leaf, 1 heart
Phase is individually insulated magnaporthe grisea spore suspension prepared by the aforementioned soil dilution planting of spray inoculation, and inoculum concentration is with rice blast
Bacterium spore suspension does not trickle on blade to be limited, and inoculation, which is placed in artificial climate incubator, to be managed, simulation 7~August part
Temperature condition observes rice leaf incidence.
Compared with prior art, biocontrol bacterial strain of the invention has preferable control effect to inducing paddy rice blast resisting, leads to
Cross soil dilution planting, seed pelleting processing and the experiment of live leaves method, the results showed that bacillus megaterium is to inducing paddy rice
The control effect of blast resisting reaches 71.97%.
The biocontrol bacterial strain category biological agent of the present invention, will not pollute environment and ecology, be conducive to the nothing of rice
Public hazards produce.
Description of the drawings
Fig. 1 is bacterial strain DdN5-6 aspect graphs.
The 16S rDNA sequential systems development tree of Fig. 2 bacterial strains DdN5-6.
Specific implementation mode
With reference to specific embodiment, the invention will be further described, in the illustrative examples and explanation of the invention
For explaining the present invention, but it is not as a limitation of the invention.
Biocontrol bacterial strain is bacillus megaterium (Bacillus megaterium) DdN5-6, and preserving number is：
CGMCCNo.15457。
Embodiment 1
Edaphon separation and indicator strain are carried out using soil dilution planting：Choose the multiple rice blast hairs in Liaoning Province
The rice terrace soil sample of sick different rice main producing region, successively dilution prepare 10-3、10-4、10-5、10-6Soil dilution liquid, by soil
Dilution takes 0.5mL to be applied to respectively in NA culture medium flat plates respectively, and each 3 ware of concentration culture is placed in 28 DEG C of insulating boxs and trains
It supports and obtains magnaporthe grisea spore suspension；After cultivating 3~4d on NA culture medium flat plates, to the bacterium grown on NA culture medium flat plates
Capable separation is dropped into, the different single bacterium colony scribed line culture of the cultural colonies such as picking form, color, purifying are then transferred to NA trainings
It supports to be put into 4 DEG C of refrigerators after being further cultured for 5d in base inclined-plane and save backup, instruction biocontrol bacterial strain is bacillus megaterium (Bacillus
megaterium)；The biocontrol microorganisms are extracted with the bacterial genomes DNA extraction kit of TIANGEN Biotech (Beijing) Co., Ltd.
Strain DNA, using bacterial 16 S rRNA gene universal primer 27F (5 '-AGAGTTTGATCCTGGTCAGAACGAACGCT-3 ') and
1492R (5 '-TACGGCTACCTTGTTACGACTTCACCCC-3 ') carries out PCR amplification, PCR reactions to the genomic DNA of extraction
Condition：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 56 DEG C of annealing 30s, 72 DEG C of renaturation 1min, repeat 30 cycles, and then 72
DEG C extend 5min, gained PCR product by Shanghai bioengineering limited liability company carry out sequencing, the sequence measured is through NCBI
After carrying out BLAST comparisons, sequence analysis is carried out with MEGA7 softwares, phylogenetic tree construction is as shown in Fig. 2, final determine instruction
Biocontrol bacterial strain is bacillus megaterium (Bacillus megaterium) DdN5-6, and biocontrol bacterial strain DdN5-6 is carried out biological guarantor
It hides, depositary institution：China Microbiological bacterial strain preservation administration committee common micro-organisms center, referred to as：CGMCC, preservation date：
2018-03-16, preserving number：CGMCCNo.15457, preservation address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese science
Institute of microbiology of institute.Bacterial strain DdN5-6 aspect graphs to bacterial strain DdN5-6 as shown in Figure 1, and carry out physio-biochemical characteristics inspection
It surveys, specifically includes：
Oxydase reaction
A filter paper is put in clean culture dish, dimethyl only makes filter paper moisten 1% aqueous solution of penylene diamines in drop
, can not overly moist.18 are taken with toothpick~lawn for 24 hours, be applied on the filter paper of moistening, the lawn smeared in 10s is existing red
Color person is the positive, and it is deferred reaction that 10s~60s, which shows red person, and 60s or more shows red person and disregards, by negative processing.
Contact enzyme reaction
The bacterium that will be cultivated for 24 hours takes a small ring to be applied to and has dripped on the slide for having 3% hydrogen peroxide with oese, if any
It is then the positive that bubble, which generates, and bubble-free is feminine gender.
Glucose oxidative fermentation reacts
With method peptone 2g, the NaCl 5g, K2HPO40.2g, glucose 10g stopped with the gloomy Er Shi culture mediums of sharp husband, fine jade
Fat 20g, bromthymol blue 1% aqueous solution 3ml, distilled water 1000ml, pH 7.0~7.2 dispense test tube, and culture medium height is about
4.5cm.121 DEG C of steam sterilization 20min.With 18~for 24 hours young age strain make seed, percutaneous puncture-inoculation, 4 every plant, wherein 2 draw and go out
The vaseline paraffin oil seal cover of bacterium, about 0.5~1cm, to completely cut off air as stopped pipe, another 2 not oil sealings are open pipe, while also to be had
The stopped pipe and open pipe not being inoculated with compare.Result is observed in thermophilic culture 1,2,3,7,14d.The only sour flavescence person of open pipe production is oxidation
Type；Open pipe and close the border that produce sour flavescence person be fermented type.
Starch Hydrolysis reacts
In gravy peptone plus 0.2% soluble starch, packing triangular flask, 121 DEG C of steam sterilization 20min are down flat plate, take new
Fresh bacterial cultures is inoculated in tablet, and after forming apparent bacterium colony, iodine solution tablet is added dropwise in indigo plant in thermophilic 2~5d of culture on tablet
Black, for periphery of bacterial colonies if any non-discolouring transparent circle, the expression Starch Hydrolysis positive is still black-and-blue for feminine gender.
V-P measures reaction
Culture medium：Peptone 5g, glucose 5g, distilled water 1000ml, pH 7.0~7.2.Reagent：Creatine 3%, NaOH
40%, strains tested is inoculated in the above culture solution, 28 DEG C of culture 4d.Culture solution and 40% mixed in equal amounts are taken, is added few
Perhaps there is red, as positive reaction in creatine, 10min such as culture solutions, it is sometimes desirable to which placing the longer time just will appear red
Reaction.
Sugar, alcoholic fermentation reaction
Gemma bacterium culture medium (NH4) 2HPO4 1.0g, KCl 0.2g, MgSO40.2g, yeast extract 0.2g, 5~6g of agar,
Sugar or alcohols 10.0g, distilled water 1000ml, 0.04% bromocresol purple 15ml.PH 7.0~7.2 dispenses test tube, culture medium height
About 4.5cm.121 DEG C of steam sterilization 20min.With 18~for 24 hours young age strain make seed, percutaneous puncture-inoculation, 4 every plant, thermophilic culture
It is observed after 1,3,5d, if indicator turns yellow, production acid is indicated, for the positive；Constant or change blue (purple) is then into feminine gender.
Aesculin hydrolysis
0.1% aesculin and 0.05% ironic citrate are added in plain broth peptone culture medium.It is oblique to dispense test tube pendulum
Face.121 DEG C of steam sterilizing 20min.It after taking fresh strain to be inoculated with, is observed after thermophilic culture 3,7,14d, it is sun to produce dark brown pigment person
Property, it is feminine gender not produce dark brown pigment person.
Clark and Lubsreaction
Culture medium：Peptone 5g, glucose 5g, NaCl 5g, water 1000ml, pH7.0~7.2, often pipe dispense 4~5ml,
121 DEG C of sterilizing 30min.Reagent：Methyl red 0.1g, 95% ethyl alcohol 300ml, distilled water 200ml, with 18~for 24 hours young age strain make
Seed is inoculated in culture solution, every time three repetitions, sets thermophilic culture 2,6d (as incubation time can be appropriately extended in feminine gender).
A drop methyl red reagent is added in culture solution, red is methyl red test positive reaction, and yellow is negative reaction (because methyl red becomes
Color range 4.4 is red to 6.0 Huangs).
Cellulose decomposition reacts
Peptone water basal medium：Peptone 5g, NaCl 5g, distilled water 1000ml, pH 7.0~7.2, packing test tube, 121
DEG C steam sterilization 20min.Basal medium is dispensed into test tube, impregnates a high-quality filter paper in the medium.Paper slip length about 5~
7cm when measuring aerobic, should have part paper slip to be exposed to outside culture medium liquid level, and when measuring anaerobism, paper slip answers complete immersion in culture medium
In, inoculation medium should have the blank control not being inoculated with.Thermophilic culture is observed for 1~4 week.Filter paper item can be resolved into one by observation
Group fiber or by filter paper item fracture or thinning person be the positive, the unchanged person of filter paper item be feminine gender.
Carbohydrate fermentation
Culture medium (Dye)：(NH4) 2HPO41.0g, KCl 0.2g, MgSO40.2g, yeast extract 0.2g, suitable carbon aquation
Close object (lactose, glucose, sucrose, mannose, maltose, mono- fructose of D, mono- xyloses of D, inositol, sorbierite, mannitol) 10g, fine jade
Fat 6g, bromocresol purple (0.04%) 15ml, distilled water 1000ml, PH7.0~7.2.It is connect with the puncture of young age bacterial strain slant culture
Kind is cultivated at above-mentioned culture medium, 28 DEG C.The first and third, it is observed after five days, if indicator turns yellow, indicates production acid, be positive anti-
It answers；Constant or bluish violet is negative reaction.
Salt tolerant is grown
Culture medium：Beef extract 3g, peptone 10g, be added various concentration NaCl (2%, 5%, 7% and 10%), culture solution
It extremely to clarify, take young age strain inoculated and cultured, cultivate 3d and 7d, be compared with nonvaccinated control tube, estimate growing state.
Gelatin liquefaction
Culture medium：Peptone 5g, gelatin 120g, water 1000ml, PH7.2~7.4, packing test tube, culture medium height about 4~
5cm, 121 DEG C of sterilizing 15min.It takes 18~culture percutaneous puncture-inoculation for 24 hours, and has two nonvaccinated blank controls, in 20 DEG C
Whether incubator culture, 2,7,10,14 and 30d liquefy in 20 DEG C of room temperature observation growing states and gelatin below, as bacterium has given birth to
Long, gelatin surface is without the grumeleuse for being recessed and stablizing, then for gelatin hydrolysis feminine gender such as gelatin clot parts or all at 20 DEG C or less
Become flowable liquid, is then gelatin liquefaction positive.
The utilization of citrate
Culture medium：NaCl 1.0g, MgSO47H2O 0.2g, NH4H2PO40.5g, sodium citrate 2g, distilled water
1000ml, 0.04% phenol red liquid 20ml, the streak inoculation on inclined-plane, thermophilic 3~7d of culture, culture medium are that (indicator is blue for alkalinity
Color or pink) person be the positive, be otherwise feminine gender.
Indoles
Culture medium：1% tryptone aqueous solution；PH 7.2~7.6 is adjusted, 1/3~1/4 test tube, 121 DEG C of steam sterilizations are dispensed
30min is inoculated in young age strain in appeal culture medium, in thermophilic culture.Reagent：Paradimethylaminobenzaldehyde 8g, ethyl alcohol
(95%) 760ml, dense HCl 160ml.The culture solution of culture 1,2,4,7d, the reagent of 3~5mm high is slowly added into training along wall pipe
At liquid layer interface red occurs for nutrient solution surface, otherwise as positive reaction is feminine gender.If color unobvious, 4~5 drops can be added
Ether shakes to culture solution, ether is made to be scattered in liquid, and culture solution is stood a moment, is adding indoles after ether to liquid level
Reagent.When as having indoles in culture solution, indoles can be extracted in ether layer, and indoles and the reagent reaction of concentration, then color is bright
It is aobvious.
Table specific as follows, the results showed that catalase, Starch Hydrolysis, sugar, alcoholic fermentation, aesculin hydrolysis, clark and Lubsreaction,
Glucose, sucrose, D-Fructose, maltose, mannitol, gelatin liquefaction and the utilization of citrate are positive；Oxidizing ferment, glucose
Oxidative fermentation, V-P measurement, cellulose decomposition, lactose, D- xyloses, mannose, inositol, sorbierite and indole reaction are negative；
The NaCl of four kinds of concentration can be grown.
Bacterial strain DdN5-6 physio-biochemical characteristics
Embodiment 2
A kind of application of the biocontrol bacterial strain of inducing paddy rice blast resisting in terms of the prevention of inducing paddy rice blast resisting, specifically
Including：
1) seed treatment：The biocontrol bacterial strain that separation obtains is transferred on NA culture medium flat plates and activates 72h at 28 DEG C, so
After be put into NB culture mediums, at 28 DEG C, 180rmin-1Shaker fermentation 3d, obtain isolated strains zymotic fluid, by 1:20 ratio
Example is to rice paddy seed difference Cotton seeds 2d；
2) live body Seedling Inoculation：It takes the rice paddy seed after aforementioned processing to carry out indoor pot, waits for that rice seedlings are grown to 3 leaf, 1 heart
Phase is individually insulated magnaporthe grisea spore suspension prepared by the aforementioned soil dilution planting of spray inoculation, and inoculum concentration is with rice blast
Bacterium spore suspension does not trickle on blade to be limited, and inoculation, which is placed in artificial climate incubator, to be managed, simulation 7~August part
Temperature condition observes rice leaf incidence.
Rice leaf incidence is counted：The disease index of untreated rice paddy seed, rice is 55.86,
The disease index of the rice paddy seed handled through DdN5-6, rice is 15.66, control effect 71.97%.
Summary, biocontrol bacterial strain of the present invention have preferable control effect to inducing paddy rice blast resisting.
Technical scheme of the present invention is not limited to the limitation of above-mentioned specific embodiment, every to do according to the technique and scheme of the present invention
The technology deformation gone out, each falls within protection scope of the present invention.
Claims (4)
1. a kind of biocontrol bacterial strain of inducing paddy rice blast resisting, it is characterised in that：The biocontrol bacterial strain is bacillus megaterium
(Bacillus megaterium) DdN5-6, preserving number are：CGMCCNo.15457.
2. the biocontrol bacterial strain of inducing paddy rice blast resisting according to claim 1, it is characterised in that：The biocontrol bacterial strain
Culture medium is NA culture mediums, and the preference temperature of biocontrol bacterial strain mycelia growth is 28 DEG C.
3. a kind of screening technique of the biocontrol bacterial strain of inducing paddy rice blast resisting as described in claim 1, which is characterized in that packet
Include following steps：
Edaphon separation and indicator strain are carried out using soil dilution planting：Choose the multiple rice blast morbidities in Liaoning Province not
The rice terrace soil sample of same rice main producing region, successively dilution prepare 10-3、10-4、10-5、10-6Soil dilution liquid, by soil dilution
Liquid takes 0.5mL to be applied to respectively in NA culture medium flat plates respectively, and each 3 ware of concentration culture is placed in 28 DEG C of insulating boxs and cultivates
To magnaporthe grisea spore suspension；After cultivating 3~4d on NA culture medium flat plates, to the bacterium colony that is grown on NA culture medium flat plates into
Row separation, the different single bacterium colony scribed line culture of the cultural colonies such as picking form, color, purifying, is then transferred to NA culture mediums
It is further cultured for being put into 4 DEG C of refrigerators after 5d in inclined-plane saving backup；Instruction biocontrol bacterial strain is bacillus megaterium (Bacillus
megaterium)。
4. a kind of biocontrol bacterial strain of inducing paddy rice blast resisting as claimed in claim 3 is in the prevention of inducing paddy rice blast resisting
The application of aspect, which is characterized in that including：
1) seed treatment：The biocontrol bacterial strain that separation obtains is transferred on NA culture medium flat plates and activates 72h at 28 DEG C, is then put
Enter in NB culture mediums, at 28 DEG C, 180rmin-1Shaker fermentation 3d, obtain isolated strains zymotic fluid, by 1:20 ratio pair
Rice paddy seed distinguishes Cotton seeds 2d；
2) live body Seedling Inoculation：It takes the rice paddy seed after aforementioned processing to carry out indoor pot, waits for that rice seedlings were grown to 1 heart stage of 3 leaf, point
Not Ge Li the magnaporthe grisea spore suspension for preparing of the aforementioned soil dilution planting of spray inoculation, inoculum concentration is with magnaporthe grisea spore
Suspension does not trickle on blade to be limited, and inoculation, which is placed in artificial climate incubator, to be managed, and the temperature item of 7~August part is simulated
Part observes rice leaf incidence.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109593676A (en) * | 2018-12-21 | 2019-04-09 | 江苏大学 | A kind of culture medium and preparation method thereof separated for microorganism in sauerkraut fermentation liquid |
-
2018
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109593676A (en) * | 2018-12-21 | 2019-04-09 | 江苏大学 | A kind of culture medium and preparation method thereof separated for microorganism in sauerkraut fermentation liquid |
| CN109593676B (en) * | 2018-12-21 | 2023-04-07 | 江苏大学 | Culture medium for separating microorganisms from pickled Chinese cabbage fermentation liquor and preparation method thereof |
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Application publication date: 20180907 |