CN105316266A - Bacillus amyloliquefaciens LX-J1 and application thereof - Google Patents
Bacillus amyloliquefaciens LX-J1 and application thereof Download PDFInfo
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Abstract
The invention relates to Bacillus amyloliquefaciens LX-J1 and application thereof. A preparation of the Bacillus amyloliquefaciens LX-J1 is prepared from a fermentation solution, copper sulfate, a phosphorus compound, a potassium compound and xanthan gum. Compared with the prior art, the liquid preparation of Bacillus amyloliquefaciens LX-J1 disclosed by the invention has the advantages that the inhibition ratio for rhizoctonia cerealis is up to 87.5%, the inhibition ratio for rhizoctonia solani is up to 78.82%, the inhibition ratio for pythium ultinum trow is up to 87%, the inhibition ratio for sclerotium rolfsii is up to 80.67%, the inhibition ratio for potato black scurf is up to 78.57%, and the inhibition ratio for corticium rolfsii saccardo can be up to more than 83%.
Description
[technical field]
The present invention relates to and belong to microbial technology field.More specifically, the present invention relates to a bacillus amyloliquefaciens LX-J1, also relate to the purposes of described bacillus amyloliquefaciens LX-J1.
[background technology]
Peanut is leguminous crop, and have another name called " Semen arachidis hypogaeae " or " peanut ", oleaginousness is up to 50%.One of main oil plant cash crop.Peanut sclerotium rolfsii is one of important disease occurred on peanut, after root, pod and basal part of stem are infected, just in the soft rotten shape of brown, flower usually first forms white spun silk at subaerial basal part of stem and the soil surface near it, and have Semen Brassicae campestris shape sclerotium, cauline leaf turns yellow, withered gradually, and peanut pod rots.This germ starts to sprout under hot and humid condition, infecting peanut, and sand, continuous continuous cropping, density are excessive stuffy, and overcast and rainy generation is heavier.The current prophylactico-therapeutic measures to peanut sclerotium rolfsii is except preferred seed and crop rotation, and the medicament of use is all chemical pesticide, and these chemical agents not only destroy ecotope and easily remain.Along with the development of science and the raising of people's living standard, it is also proposed higher requirement to food safety and environmental protection agricultural, therefore new and effective biological pesticide and fertilizer are controlling plant diseases will be the inexorable trend of Organic Farming.
Bacillus amyloliquefaciens has fast, adaptable, good stability, the metabolic way feature widely of growth; There is Promoting plant growth, with the characteristic suppressing native transmissibility pathogenic bacteria, and very strong resistance can be had to heat, electromagnetic radiation, ultraviolet and some pharmaceutical chemicalss, exactly because have so strong resistance, the formulation making it be conducive to biocontrol fungicide is produced, and therefore bacillus amyloliquefaciens is the biocontrol microorganisms as agricultural microorganism with higher Development volue.
[summary of the invention]
[technical problem that will solve]
The object of this invention is to provide a bacillus amyloliquefaciens LX-J1.
Another object of the present invention is to provide the purposes of described bacillus amyloliquefaciens LX-J1.
[technical scheme]
The present invention is achieved through the following technical solutions.
The present invention relates to a bacillus amyloliquefaciens (Bacillusamyloliquefaciens) LX-J1, this bacterial strain is on the August 21st, 2015 of No. 3 China Committee for Culture Collection of Microorganisms of Institute of Microorganism, Academia Sinica common micro-organisms center preservations in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and its preserving number is CGMCCNo.11263.
A preferred embodiment of the invention, the step of the cultural method of described bacillus amyloliquefaciens LX-J1 is as follows:
A, slant culture
Use the transfering loop picking one environmental protection bacillus amyloliquefaciens LX-J1 be stored in glycerine pipe to be inoculated on slant medium inclined-plane, be then placed in constant incubator and cultivate 40 ~ 48 hours at the condition lower inclined plane of temperature 28 ~ 30 DEG C;
B, seed culture
One ring is inoculated in liquid seed culture medium at the slant culture that steps A obtains, is then placed in constant incubator and cultivates 6h ~ 10h at temperature 28 ~ 30 DEG C with the condition of rotating speed 180rpm, obtain inoculum;
C, fermentation culture
According to fermention medium volumeter 5 ~ 10% inoculum size, the inoculum obtained in step B is forwarded in fermention medium, be placed in fermentation culture tank again, first under temperature 28 ~ 30 DEG C, rotating speed 150 ~ 200rpm, air quantity 1:1.0 ~ 1.2 and the condition of tank internal pressure 0.04 ~ 0.08MPa, cultivate 15h, then at temperature 30 ~ 35 DEG C with the condition of rotating speed 320 ~ 380rpm, continue fermentation culture to 40-45h, the fermenting culture obtained after fermentation ends contains 100 ~ 12,000,000,000/ml bacillus amyloliquefaciens LX-J1.
According to another kind of preferred implementation of the present invention, the preparation method of described slant medium is as follows:
By soluble in water to 5 ~ 10g extractum carnis, 1 ~ 5g sodium-chlor, 5 ~ 10g peptone and 20g agar, then add to cumulative volume 1000ml with water, then use mineral acid or mineral alkali that the solution ph obtained is adjusted to pH7.0 ~ 7.2.
According to another kind of preferred implementation of the present invention, the preparation method of described liquid seed culture medium is as follows:
By soluble in water to 5 ~ 10g extractum carnis, 1 ~ 5g sodium-chlor and 5 ~ 10g peptone, then add to cumulative volume 1000ml with water, then use mineral acid or mineral alkali that the solution ph obtained is adjusted to pH7.0 ~ 7.2.
According to another kind of preferred implementation of the present invention, the preparation method of described fermention medium is as follows:
By 15 ~ 20g starch, 1 ~ 3g corn starch, 1 ~ 5g peptone, 1 ~ 5g sodium-chlor, 1 ~ 5gK
2hPO
43H
2o, 1 ~ 3gNH
4cl and 0.1 ~ 0.5gMgSO
47H
2o is soluble in water, then adds to cumulative volume 1000ml with water, then uses mineral acid or mineral alkali that the solution ph obtained is adjusted to pH7.0 ~ 7.5.
According to another kind of preferred implementation of the present invention, described mineral acid is selected from hydrochloric acid or phosphoric acid; Described mineral alkali is selected from sodium hydroxide, potassium hydroxide or ammoniacal liquor.
The invention still further relates to a kind of production method of bacillus amyloliquefaciens LX-J1 preparation.
The step of this production method is as follows:
Add in described bacillus amyloliquefaciens LX-J1 fermented liquid with metal ion weight count 0.1 ~ 0.5% copper sulfate, the zinc sulfate of 1 ~ 2%, the manganous sulfate of 0.1 ~ 0.5% and 0.1 ~ 0.6% xanthan gum, obtain described bacillus amyloliquefaciens LX-J1 preparation.
The invention still further relates to and adopt described production method to produce the bacillus amyloliquefaciens LX-J1 preparation obtained.Every milliliter of bacillus amyloliquefaciens LX-J1 preparation contained 10,000,000,000 with last bacillus amyloliquefaciens LX-J1.
The invention still further relates to the purposes of described bacillus amyloliquefaciens LX-J1 preparation in control Rhizoctonia cerealis, dry thread Pyrenomycetes, Pythium ultimum, sclerotium rolfsii and the black hemorrhoid disease of potato.
In more detail the present invention will be described below.
The present invention relates to a bacillus amyloliquefaciens (Bacillusamyloliquefaciens) LX-J1, this bacterial strain is on the August 21st, 2015 of No. 3 China Committee for Culture Collection of Microorganisms of Institute of Microorganism, Academia Sinica common micro-organisms center preservations in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and its preserving number is CGMCCNo.11263.
One, separation screening
Gather pedotheque from Zhumadian peanut cultivation plot, Henan, within the scope of the degree of depth 10 ~ 20cm, take 100g soil in plot surrounding and central authorities respectively according to five point samplings, these soil equivalent is mixed, obtains a kind of soil sample.Taking 10g soil sample is dissolved in 90ml water, and vibrate 20min in shaking culture case at temperature 28 DEG C with the condition of rotating speed 180rpm, and being separated and obtaining concentration is 10
-2the suspension of g/ml.
Pipette 100 μ l suspension to be put in the centrifuge tube that 900 μ l water are housed, mixing of fully vibrating, makes 10
-3the suspension of g/ml, then be diluted with water to 10 successively
-7or 10
-8g/ml suspension.Pipette 100 μ l10 respectively
-7or 10
-8g/ml suspension, PDA solid medium (20g glucose, 200g peeled potatoes, 18g agar powder and 1000ml distilled water are uniformly coated on, pH7.0) in flat board, on flat board, inoculate a Sclerotium rolfssi sclerotium according to each plate center simultaneously, then be put in temperature 25 DEG C of incubators to be inverted and cultivate 4d, observe and select antagonism and suppress the bacterium colony that circle is large.
Two, purifying
Allow the bacterium colony selected in step one at NA substratum (peptone 10g/L, extractum carnis 5g/L, sodium-chlor 5g/L, agar 20g/L) on again to rule purifying, picking list bacterium colony point is inoculated on above-mentioned PDA substratum, substratum is inoculated Sclerotium rolfssi and carries out antagonism checking, and picking has single colony inoculation of identical colonial morphology on NA substratum, line is preserved.
Three, identification of strains
1. thalli morphology characteristic: gram-positive microorganism, shaft-like, NA substratum is cultivated more than 12h and can produce gemma, specifically see accompanying drawing 1.
2. colonial morphology characteristic: when being cultured to bacterium colony 3mm on NA substratum colonial morphology for circular, have projection, there is fold on surface, edge is wavy opaque white color bacterium colony, specifically see accompanying drawing 2.
3. molecular biology identification (16srDNA sequence amplification and sequencing):
Prepared by sample total DNA: adopt Conventional bacteria DNA extraction method to carry out.
PCR primer: forward primer 27F:AGAGTTTGATCMTGGCTCAG; Reverse primer 1492r:TACGGYTACCTTGTTACGACTT.
PCR reaction system: 20 μ l reaction systems, reaction solution forms: 1 μ lDNA template, 10 μ l2 × MastarMix, 0.4 μ l27F, 0.4 μ l1492R, ultrapure water complements to 20 μ l.
Pcr amplification program: be 2min temperature 94 DEG C; 30 circulations (be 1min temperature 94 DEG C, being 1min temperature 52 DEG C, is 8min temperature 65 DEG C); Be 18min temperature 65 DEG C.
Amplified production detects order-checking correctly through 0.7% agarose gel electrophoresis, sequencing result utilizes blast software to carry out sequence analysis, the homology of this bacterial strain and bacillus amyloliquefaciens is 99%, in conjunction with colony characteristics, morphological features and molecular biological characteristic, identify that this isolate is bacillus amyloliquefaciens (Bacillusamyloliquefaciens).The 16SrRNA gene sequencing of bacillus amyloliquefaciens CGMCCNo.11263 bacterial strain the results are shown in annex 1.
This Strain Designation is bacillus amyloliquefaciens LX-J1, it on August 21st, 2015 in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, its preserving number is CGMCCNo.11263.
According to the present invention, the step of the cultural method of described bacillus amyloliquefaciens LX-J1 is as follows:
A, slant culture
Use the transfering loop picking one environmental protection bacillus amyloliquefaciens LX-J1 be stored in glycerine pipe to be inoculated on slant medium inclined-plane, be then placed in constant incubator and cultivate 40 ~ 48 hours at the condition lower inclined plane of temperature 28 ~ 30 DEG C.
The preparation method of described slant medium is as follows:
By soluble in water to 5 ~ 10g extractum carnis, 1 ~ 5g sodium-chlor, 5 ~ 10g peptone and 20g agar, then add to cumulative volume 1000ml with water, then use mineral acid or mineral alkali that the solution ph obtained is adjusted to pH7.0 ~ 7.2.Described slant medium is for its slant culture after the sterilizing of employing conventional sterilization procedures.
Described mineral acid is selected from hydrochloric acid or phosphoric acid; Described mineral alkali is selected from sodium hydroxide, potassium hydroxide or ammoniacal liquor.
Mineral acid used in the present invention is inorganic acid aqueous solution, and mineral alkali is inorganic base aqueous solution.Described mineral acid or the concentration of inorganic base aqueous solution normally 0.1 ~ 2.0N.
The mineral acid used at subsequent step of the present invention or mineral alkali situation identical as previously described, therefore repeat no more.
Preferably, described slant medium is prepared by 6 ~ 9g extractum carnis, 1.8 ~ 4.2g sodium-chlor, 6 ~ 9g peptone and 20g agar.
More preferably, described slant medium is prepared by 7 ~ 8g extractum carnis, 2.5 ~ 3.4g sodium-chlor, 7 ~ 8g peptone and 20g agar.
In the present invention, the raw material used when preparing slant medium is all product sold in the market.
Constant incubator used in this step is product sold in the market, " two five metals " the board DHP120 constant incubator such as produced by Shanghai laboratory apparatus Co., Ltd., Factory.
B, seed culture
One ring is inoculated in liquid seed culture medium at the slant culture that steps A obtains, is then placed in constant incubator and cultivates 6h ~ 10h at temperature 28 ~ 30 DEG C with the condition of rotating speed 180rpm, obtain inoculum;
The preparation method of described liquid seed culture medium is as follows:
By soluble in water to 5 ~ 10g extractum carnis, 1 ~ 5g sodium-chlor and 5 ~ 10g peptone, then add to cumulative volume 1000ml with water, then use mineral acid or mineral alkali that the solution ph obtained is adjusted to pH7.0 ~ 7.2.Described liquid seed culture medium is for its seed culture after the sterilizing of employing conventional sterilization procedures.
Preferably, described liquid seed culture medium is prepared by 6 ~ 9g extractum carnis, 1.8 ~ 4.2g sodium-chlor and 6 ~ 9g peptone.
More preferably, described liquid seed culture medium is prepared by 7 ~ 8g extractum carnis, 2.4 ~ 3.6g sodium-chlor and 7 ~ 8g peptone.
In the present invention, the raw material used when preparing liquid seed culture medium is all product sold in the market.
Constant incubator used in this step is product sold in the market, the QHZ-98A type total temperature shaking culture case such as produced by the magnificent biochemical instrument factory in Taicang.
The bacillus amyloliquefaciens LX-J1 content of described inoculum adopts viable plate counts conventional in the art to measure.
C, fermentation culture
According to fermention medium volumeter 5 ~ 10% inoculum size, the inoculum obtained in step B is forwarded in fermention medium, be placed in fermentation culture tank again, first under temperature 28 ~ 30 DEG C, rotating speed 150 ~ 200rpm, air quantity 1:1.0 ~ 1.2 and the condition of tank internal pressure 0.04 ~ 0.08MPa, cultivate 15h, then at temperature 30 ~ 35 DEG C with the condition of rotating speed 320 ~ 380rpm, continue fermentation culture to 40-45h, the fermenting culture obtained after fermentation ends contains 100 ~ 12,000,000,000/ml bacillus amyloliquefaciens LX-J1.
The preparation method of described fermention medium is as follows:
By 15 ~ 20g starch, 1 ~ 3g corn starch, 1 ~ 5g peptone, 1 ~ 5g sodium-chlor, 1 ~ 5gK
2hPO
43H
2o, 1 ~ 3gNH
4cl and 0.1 ~ 0.5gMgSO
47H
2o is soluble in water, then adds to cumulative volume 1000ml with water, then uses mineral acid or mineral alkali that the solution ph obtained is adjusted to pH7.0 ~ 7.5.
Preferably, described fermention medium is by 16 ~ 20g starch, 1.6 ~ 2.6g corn starch, 1.8 ~ 4.2g peptone, 1.5 ~ 4.5g sodium-chlor, 1.5 ~ 4.5gK
2hPO
43H
2o, 1.4 ~ 2.6gNH
4cl and 0.1 ~ 0.4gMgSO
47H
2o prepares.
More preferably, described fermention medium is by 18 ~ 20g starch, 1.8 ~ 2.4g corn starch, 2.6 ~ 3.6g peptone, 2.5 ~ 3.5g sodium-chlor, 2.5 ~ 3.4gK
2hPO
43H
2o, 1.6 ~ 2.4gNH
4cl and 0.2 ~ 0.4gMgSO
47H
2o prepares.
In the present invention, described air quantity should be appreciated that it is the air flow of unit time, and such as, in fermentor tank, nutrient solution volume is 50L, and air quantity 1:1.0 ~ 1.2 represent that the air flow in fermentor tank is 50-60L/h.
In the present invention, the raw material used when preparing fermention medium is all product sold in the market.
The fermentation culture tank that the present invention uses is product sold in the market, the FUS-50L type fermentor tank such as produced by Guoqiang's biochemical engineering Equipment Limited.
The invention still further relates to a kind of production method of bacillus amyloliquefaciens LX-J1 preparation.
The step of this production method is as follows:
Add in described bacillus amyloliquefaciens LX-J1 fermented liquid with metal ion weight count 0.1 ~ 0.5% copper sulfate, the zinc sulfate of 1 ~ 2%, the manganous sulfate of 0.1 ~ 0.5% and 0.1 ~ 0.6% xanthan gum, obtain described bacillus amyloliquefaciens LX-J1 preparation.
The effect adding copper sulfate, zinc sulfate and manganous sulfate in the present invention in described fermented liquid is to provide trace element needed for crop.
In the present invention, production cost can be increased when copper sulfate, zinc sulfate and manganous sulfate addition exceed described scope and easily can produce precipitation and cause and use inconvenience.
The effect adding xanthan gum in fermented liquid has been dissemination, makes solution be even shape.
The invention still further relates to and adopt described production method to produce the bacillus amyloliquefaciens LX-J1 preparation obtained.Every milliliter of bacillus amyloliquefaciens LX-J1 preparation contained 10,000,000,000 with last bacillus amyloliquefaciens LX-J1.
The invention still further relates to described bacillus amyloliquefaciens LX-J1 preparation to Rhizoctonia cerealis, dry thread Pyrenomycetes, all have antagonistic action to Pythium ultimum, the black hemorrhoid disease of potato, therefore described preparation may be used for control Rhizoctonia cerealis, dry thread Pyrenomycetes, Pythium ultimum, sclerotium rolfsii hemorrhoid disease black in potato.
Adopt the pathogenic bacteria of dull and stereotyped face-off method to the black hemorrhoid disease of Rhizoctonia cerealis, dry thread Pyrenomycetes, Pythium ultimum, sclerotium rolfsii and potato to carry out antagonistic experiment, result show bacillus amyloliquefaciens LX-J1 liquid preparation of the present invention to the inhibiting rate of Rhizoctonia cerealis be 87.5%, the inhibiting rate of Rhizoctonia solani is 78.82%, be 87% to the inhibiting rate of Pythium ultimum, be 80.67% to the inhibiting rate of sclerotium rolfsii, be 78.57% to the inhibiting rate of the black hemorrhoid disease of potato.
According to the ratio of 1:50 (w/w), use bacillus amyloliquefaciens LX-J1 preparation of the present invention to dress seed to peanut, plant peanut test determine, bacillus amyloliquefaciens LX-J1 liquid preparation of the present invention can reach more than 83% to the preventive effect of peanut sclerotium rolfsii.
[beneficial effect]
The invention has the beneficial effects as follows: compared with prior art, bacillus amyloliquefaciens LX-J1 liquid preparation of the present invention to the inhibiting rate of Rhizoctonia cerealis be 87.5%, the inhibiting rate of Rhizoctonia solani is 78.82%, be 87% to the inhibiting rate of Pythium ultimum, be 80.67% to the inhibiting rate of sclerotium rolfsii, be 78.57% to the inhibiting rate of the black hemorrhoid disease of potato, can more than 83% be reached to the preventive effect of peanut sclerotium rolfsii.
[accompanying drawing explanation]
Fig. 1 is the micro-kenel figure of bacillus amyloliquefaciens LX-J1;
Fig. 2 is the colonial morphology figure of bacillus amyloliquefaciens LX-J1 on NA substratum;
Fig. 3 is that bacillus amyloliquefaciens LX-J1 is to cereal silk core disease pathogen bacterium antagonism figure;
Fig. 4 is that bacillus amyloliquefaciens LX-J1 is to miliary damping-off disease pathogen bacterium antagonism figure;
Fig. 5 is that bacillus amyloliquefaciens LX-J1 is to Pythium ultimum pathogenic bacteria antagonism figure;
Fig. 6 is bacillus amyloliquefaciens LX-J1 dialogue thin,tough silk disease pathogen bacterium antagonism figure;
Fig. 7 is that bacillus amyloliquefaciens LX-J1 is to potato black hemorrhoid disease pathogen bacterium antagonism figure;
[embodiment]
The present invention can be understood better by following embodiment.
Embodiment 1: the cultivation of bacillus amyloliquefaciens LX-J1
The implementation step of this embodiment is as follows:
A, slant culture
Prepare slant medium: by soluble in water to 5g extractum carnis, 2.5g sodium-chlor, 5g peptone and 20g agar, then add to cumulative volume 1000ml with water, then use 0.8N hydrochloric acid or aqueous sodium hydroxide solution that the solution ph obtained is adjusted to pH7.0.
Use the transfering loop picking one environmental protection bacillus amyloliquefaciens LX-J1 be stored in glycerine pipe to be inoculated on slant medium inclined-plane, be then placed in constant incubator and cultivate 48 hours at the condition lower inclined plane of temperature 28 DEG C;
B, seed culture
Prepare liquid seed culture medium: by soluble in water to 7g extractum carnis, 3g sodium-chlor and 5g peptone, then add to cumulative volume 1000ml with water, then use 0.8N hydrochloric acid or aqueous sodium hydroxide solution that the solution ph obtained is adjusted to pH7.0.
One ring is inoculated in liquid seed culture medium at the slant culture that steps A obtains, is then placed in constant incubator and cultivates 5h at temperature 28 DEG C with the condition of rotating speed 180rpm, obtain inoculum;
C, fermentation culture
Prepare fermention medium: by 16g starch, 1.0g corn starch, 2.5g peptone, 2.5g sodium-chlor, 1.0gK
2hPO
43H
2o, 1.0gNH
4cl and 0.2gMgSO
47H
2o is soluble in water, then adds to cumulative volume 1000ml with water, then uses 0.1N hydrochloric acid or aqueous sodium hydroxide solution that the solution ph obtained is adjusted to pH7.2.
According to fermention medium volumeter 5% inoculum size, the inoculum obtained in step B is forwarded in fermention medium, be placed in fermentation culture tank again, first under the condition of temperature 28 DEG C, rotating speed 150rpm, air quantity 1:1.0 and tank internal pressure 0.06MPa, cultivate 15h, then at temperature 30 DEG C with the condition of rotating speed 360rpm, fermentation culture is continued to 42h, adopt viable plate counts conventional in the art to measure, the fermenting culture obtained after fermentation ends contains 10,900,000,000/ml bacillus amyloliquefaciens LX-J1.
Embodiment 2: the cultivation of bacillus amyloliquefaciens LX-J1
The implementation step of this embodiment is as follows:
A, slant culture
Prepare slant medium: by soluble in water to 7g extractum carnis, 3.4g sodium-chlor, 10g peptone and 20g agar, then add to cumulative volume 1000ml with water, then use 0.1N hydrochloric acid or potassium hydroxide aqueous solution that the solution ph obtained is adjusted to pH7.1.
Use the transfering loop picking one environmental protection bacillus amyloliquefaciens LX-J1 be stored in glycerine pipe to be inoculated on slant medium inclined-plane, be then placed in constant incubator and cultivate 40 hours at the condition lower inclined plane of temperature 29 DEG C;
B, seed culture
Prepare liquid seed culture medium: by soluble in water to 8g extractum carnis, 2.4g sodium-chlor and 10g peptone, then add to cumulative volume 1000ml with water, then use 0.1N hydrochloric acid or potassium hydroxide aqueous solution that the solution ph obtained is adjusted to pH7.1.
One ring is inoculated in liquid seed culture medium at the slant culture that steps A obtains, is then placed in constant incubator and cultivates 6h at temperature 29 DEG C with the condition of rotating speed 180rpm, obtain inoculum;
C, fermentation culture
Prepare fermention medium: by 18g starch, 1.8g corn starch, 5.0g peptone, 3.5g sodium-chlor, 2.5gK
2hPO
43H
2o, 3.0gNH
4cl and 0.4gMgSO
47H
2o is soluble in water, then adds to cumulative volume 1000ml with water, then uses 0.1N hydrochloric acid or potassium hydroxide aqueous solution that the solution ph obtained is adjusted to pH7.0.
According to fermention medium volumeter 6% inoculum size, the inoculum obtained in step B is forwarded in fermention medium, be placed in fermentation culture tank again, first under the condition of temperature 29 DEG C, rotating speed 170rpm, air quantity 1:1.1 and tank internal pressure 0.04MPa, cultivate 15h, then at temperature 32 DEG C with the condition of rotating speed 320rpm, fermentation culture is continued to 40h, adopt viable plate counts conventional in the art to measure, the fermenting culture obtained after fermentation ends contains 10,600,000,000/ml bacillus amyloliquefaciens LX-J1.
Embodiment 3: the cultivation of bacillus amyloliquefaciens LX-J1
The implementation step of this embodiment is as follows:
A, slant culture
Prepare slant medium: by soluble in water to 8g extractum carnis, 2.0g sodium-chlor, 7g peptone and 20g agar, then add to cumulative volume 1000ml with water, then use 2.0N phosphoric acid or ammonia soln that the solution ph obtained is adjusted to pH7.2.
Use the transfering loop picking one environmental protection bacillus amyloliquefaciens LX-J1 be stored in glycerine pipe to be inoculated on slant medium inclined-plane, be then placed in constant incubator and cultivate 44 hours at the condition lower inclined plane of temperature 30 DEG C;
B, seed culture
Prepare liquid seed culture medium: by soluble in water to 5g extractum carnis, 3.6g sodium-chlor and 7g peptone, then add to cumulative volume 1000ml with water, then use 2.0N phosphoric acid or ammonia soln that the solution ph obtained is adjusted to pH7.2.
One ring is inoculated in liquid seed culture medium at the slant culture that steps A obtains, is then placed in constant incubator and cultivates 10h at temperature 30 DEG C with the condition of rotating speed 180rpm, obtain inoculum;
C, fermentation culture
Prepare fermention medium: by 18g starch, 2.4g corn starch, 2.6g peptone, 1.0g sodium-chlor, 3.4gK
2hPO
43H
2o, 1.6gNH
4cl and 0.1gMgSO
47H
2o is soluble in water, then adds to cumulative volume 1000ml with water, then uses 2.0N phosphoric acid or ammonia soln that the solution ph obtained is adjusted to pH7.4.
According to fermention medium volumeter 8% inoculum size, the inoculum obtained in step B is forwarded in fermention medium, be placed in fermentation culture tank again, first under the condition of temperature 30 DEG C, rotating speed 200rpm, air quantity 1:1.2 and tank internal pressure 0.06MPa, cultivate 15h, then at temperature 34 DEG C with the condition of rotating speed 380rpm, fermentation culture is continued to 45h, adopt viable plate counts conventional in the art to measure, the fermenting culture obtained after fermentation ends contains 12,000,000,000/ml bacillus amyloliquefaciens LX-J1.
Embodiment 4: the cultivation of bacillus amyloliquefaciens LX-J1
The implementation step of this embodiment is as follows:
A, slant culture
Prepare slant medium: by soluble in water to 10g extractum carnis, 5.0g sodium-chlor, 8g peptone and 20g agar, then add to cumulative volume 1000ml with water, then use 1.4N hydrochloric acid or ammonia soln that the solution ph obtained is adjusted to pH7.2.
Use the transfering loop picking one environmental protection bacillus amyloliquefaciens LX-J1 be stored in glycerine pipe to be inoculated on slant medium inclined-plane, be then placed in constant incubator and cultivate 46 hours at the condition lower inclined plane of temperature 30 DEG C;
B, seed culture
Prepare liquid seed culture medium: by soluble in water to 10g extractum carnis, 5.0g sodium-chlor and 8g peptone, then add to cumulative volume 1000ml with water, then use 1.4N hydrochloric acid or ammoniacal liquor that the solution ph obtained is adjusted to pH7.2.
One ring is inoculated in liquid seed culture medium at the slant culture that steps A obtains, is then placed in constant incubator and cultivates 8h at temperature 30 DEG C with the condition of rotating speed 180rpm, obtain inoculum;
C, fermentation culture
Prepare fermention medium: by 20g starch, 3.0g corn starch, 3.6g peptone, 5.0g sodium-chlor, 5.0gK
2hPO
43H
2o, 2.4gNH
4cl and 0.5gMgSO
47H
2o is soluble in water, then adds to cumulative volume 1000ml with water, then uses 1.4N hydrochloric acid or ammonia soln that the solution ph obtained is adjusted to pH7.5.
According to fermention medium volumeter 10% inoculum size, the inoculum obtained in step B is forwarded in fermention medium, be placed in fermentation culture tank again, first under the condition of temperature 30 DEG C, rotating speed 200rpm, air quantity 1:1.1 and tank internal pressure 0.08MPa, cultivate 15h, then at temperature 35 DEG C with the condition of rotating speed 340rpm, fermentation culture is continued to 40h, adopt viable plate counts conventional in the art to measure, the fermenting culture obtained after fermentation ends contains 11,500,000,000/ml bacillus amyloliquefaciens LX-J1.
Embodiment 5: the production of bacillus amyloliquefaciens LX-J1 preparation
The implementation step of this embodiment is as follows:
Toward embodiment 1 prepare bacillus amyloliquefaciens LX-J1 fermented liquid in add with metal ion weight count the copper sulfate of 0.2%, the zinc sulfate of 1.0% and 0.4% manganous sulfate and 0.1% xanthan gum, obtain described bacillus amyloliquefaciens LX-J1 preparation.Adopt viable plate counts conventional in the art to measure, every milliliter of bacillus amyloliquefaciens LX-J1 preparation contains 10,300,000,000 bacillus amyloliquefaciens LX-J1.
Embodiment 6: the production of bacillus amyloliquefaciens LX-J1 preparation
The implementation step of this embodiment is as follows:
Toward embodiment 2 prepare bacillus amyloliquefaciens LX-J1 fermented liquid in add with metal ion weight count the copper sulfate of 0.1%, the zinc sulfate of 1.4% and 0.1% manganous sulfate 0.6% xanthan gum, obtain described bacillus amyloliquefaciens LX-J1 preparation.Adopt viable plate counts conventional in the art to measure, every milliliter of bacillus amyloliquefaciens LX-J1 preparation contains 10,200,000,000 bacillus amyloliquefaciens LX-J1.
Embodiment 7: the production of bacillus amyloliquefaciens LX-J1 preparation
The implementation step of this embodiment is as follows:
Add in the bacillus amyloliquefaciens LX-J1 fermented liquid of the preparation of embodiment with metal ion weight count the copper sulfate of 0.5%, the zinc sulfate of 1.8% and 0.5% manganous sulfate 0.3% xanthan gum, obtain described bacillus amyloliquefaciens LX-J1 preparation.Adopt viable plate counts conventional in the art to measure, every milliliter of bacillus amyloliquefaciens LX-J1 preparation contains 11,000,000,000 bacillus amyloliquefaciens LX-J1.
Embodiment 8: the production of bacillus amyloliquefaciens LX-J1 preparation
The implementation step of this embodiment is as follows:
Toward embodiment 4 prepare bacillus amyloliquefaciens LX-J1 fermented liquid in add with metal ion weight count the copper sulfate of 0.4%, the zinc sulfate of 2.0% and 0.3% manganous sulfate 0.5% xanthan gum, obtain described bacillus amyloliquefaciens LX-J1 preparation.Adopt viable plate counts conventional in the art to measure, every milliliter of bacillus amyloliquefaciens LX-J1 preparation contains 10,400,000,000 bacillus amyloliquefaciens LX-J1.
Embodiment 9: the dull and stereotyped antagonistic ability test of bacillus amyloliquefaciens LX-J1 preparation
Preparation PDA solid culture: according to 200g/L ratio, peeled potatoes is placed in water and boils 30min, filter with 2 layers of gauze after cooling, again according to the ratio of 20g/L sucrose and 20g/L agar, in the filtrate obtained, add sucrose and agar, after sterilizing, obtain PDA solid culture.
Be seeded in PDA solid culture by Rhizoctonia cerealis, dry thread Pyrenomycetes, Pythium ultimum bacterium, Sclerotium rolfssi, the black hemorrhoid germ of potato respectively, at temperature 25 DEG C, cultivate 4-5d, treat that pathogenic fungi covers with whole culture dish, its particular case is see accompanying drawing 3-7.
Antagonistic effect measures:
First from culture dish, get with punch tool the pathogenic bacteria bacterium cake that its diameter is about 6mm, be placed in PDA plate center, be about the punching of 150mm place respectively in the left and right sides, and add 20 μ l bacillus amyloliquefaciens preparation of the present invention; Simultaneously not add bacillus amyloliquefaciens preparation in contrast, 4-5d is cultivated temperature 25 DEG C, treat that whole culture dish is covered with in contrast, record bacillus amyloliquefaciens preparation is to the bacteriostatic diameter of each pathogenic bacteria, its test-results is listed in table 1, and obtaining corresponding bacteriostasis rate according to following formulae discovery, its result is also listed in table 1.
Table 1: bacillus amyloliquefaciens preparation bacteria inhibition assay result
Colony growth inhibiting rate=(contrast bacterium colony clean growth diameter-clean growth diameter of process bacterium colony)/clean growth diameter × 100% of contrast bacterium colony
The result of table 1 clearly illustrates that, bacillus amyloliquefaciens LX-J1 of the present invention is to Rhizoctonia cerealis, dry thread Pyrenomycetes, to the inhibition of Pythium ultimum, peanut sclerotium rolfsii, the black hemorrhoid germ of potato all clearly, bacteriostasis rate reaches 87.5%, 78.82%, 87%, 80.67%, 78.57% respectively.
Embodiment 10: bacillus amyloliquefaciens LX-J1 preparation of the present invention is to the prevention effect of peanut sclerotium rolfsii.
One, peanut pot-culture method:
Load sterile soil in every basin, loadings is every basin is 1000g.In every basin, plant the excellent peanut seed that 5 processes are selected, each process five weights, each repetition establishes three process altogether:
1, blank, is directly sprinkled into peanut seed, and covers one deck sterile soil above;
2, adopt and cover with whole mycelia in the culture dish of peanut sclerotium rolfsii pathogenic bacteria by one and scrape in 50ml sterilized water, vibrate about 30min, by the ratio of itself and peanut seed according to the amount of 1:50 at peanut seed surface seeding Sclerotium rolfssi, dry, again the seed of inoculation Sclerotium rolfssi is sprinkled in flowerpot, and covers one deck sterile soil above;
3, the amount being 1:50 with the usage ratio of bacillus amyloliquefaciens LX-J1 preparation of the present invention and peanut seed is dressed seed to peanut seed, dry, adopt again and cover with whole mycelia in the culture dish of peanut sclerotium rolfsii pathogenic bacteria by one and scrape in 50ml sterilized water, vibrate about 30min, by the ratio of itself and peanut seed according to the amount of 1:50 (w/w) at peanut seed surface seeding Sclerotium rolfssi, dry, then plant in flowerpot, and cover one deck sterile soil above;
Two, the plant of bacteria infection carries out classification according to following standard:
0 grade: plants stems base portion and main root are all without scab;
1 grade: basal part of stem and main root have 1-3 scab;
3 grades: on basal part of stem or main root, scab is more, and lesion area accounts for 1/4 ~ 1/2 of stem and the root total area;
5 grades: on basal part of stem and main root, scab is many and comparatively large, and lesion area accounts for 1/2 ~ 3/4 of basal part of stem and the root total area;
7 grades: on basal part of stem or main root, scab in flakes, formed and burn stem phenomenon, but root system is not dead;
9 grades: root system is downright bad, plant above ground portion wilts or death.
By the ranked data obtained in test, calculate disease index according to the following formula, then calculate prevention effect % according to disease index.
Three, record situation of emerging and calculate seedling rate
Observe 10-15 days situations of emerging, record seedling rate.According to the volume ratio 1:500 of described bacillus amyloliquefaciens LX-J1 preparation and water, prepare bacillus amyloliquefaciens LX-J1 inhibitor dilutions.After peanut seedling grows, within every 10 days, water the diluent described in a 300ml.To observe after 30 days and according to above-mentioned part grade standard record incidence, calculate sickness rate % according to the following formula, the results are shown in Table 2 for it.
Sickness rate %=(strain number of catching an illness/investigate total strain number) × 100%
Table 2: bacillus amyloliquefaciens LX-J1 microbial inoculum is to the prevention effect of the white thin,tough silk bacterium of peanut
| Process | Sickness rate % | Disease index % | Prevention effect % |
| 1 | 0％ | － | － |
| 2 | 70％ | 10.58 | － |
| 3 | 20％ | 1.76 | 83.36 |
The result of table 2 obviously shows, in this experiment, the preventive effect of bacillus amyloliquefaciens LX-J1 microbial inoculum to the white thin,tough silk bacterium of peanut reaches 83.36%.
Claims (9)
1. a bacillus amyloliquefaciens (Bacillusamyloliquefaciens) LX-J1, this bacterial strain is on the August 21st, 2015 of No. 3 China Committee for Culture Collection of Microorganisms of Institute of Microorganism, Academia Sinica common micro-organisms center preservations in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and its preserving number is CGMCCNo.11263.
2. the cultural method of bacillus amyloliquefaciens LX-J1 according to claim 1, is characterized in that the step of the method is as follows:
A, slant culture
Use the transfering loop picking one environmental protection bacillus amyloliquefaciens LX-J1 be stored in glycerine pipe to be inoculated on slant medium inclined-plane, be then placed in constant incubator and cultivate 40 ~ 48 hours at the condition lower inclined plane of temperature 28 ~ 30 DEG C;
B, seed culture
One ring is inoculated in liquid seed culture medium at the slant culture that steps A obtains, is then placed in constant incubator and cultivates 6h ~ 10h at temperature 28 ~ 30 DEG C with the condition of rotating speed 180rpm, obtain inoculum;
C, fermentation culture
According to fermention medium volumeter 5 ~ 10% inoculum size, the inoculum obtained in step B is forwarded in fermention medium, be placed in fermentation culture tank again, first under temperature 28 ~ 30 DEG C, rotating speed 150 ~ 200rpm, air quantity 1:1.0 ~ 1.2 and the condition of tank internal pressure 0.04 ~ 0.08MPa, cultivate 15h, then at temperature 30 ~ 35 DEG C with the condition of rotating speed 320 ~ 380rpm, continue fermentation culture to 40-45h, the fermenting culture obtained after fermentation ends contains 100 ~ 12,000,000,000/ml bacillus amyloliquefaciens LX-J1.
3. cultural method according to claim 2, is characterized in that the preparation method of described slant medium is as follows:
By soluble in water to 5 ~ 10g extractum carnis, 1 ~ 5g sodium-chlor, 5 ~ 10g peptone and 20g agar, then add to cumulative volume 1000ml with water, then use mineral acid or mineral alkali that the solution ph obtained is adjusted to pH7.0 ~ 7.2.
4. cultural method according to claim 2, is characterized in that the preparation method of described liquid seed culture medium is as follows:
By soluble in water to 5 ~ 10g extractum carnis, 1 ~ 5g sodium-chlor and 5 ~ 10g peptone, then add to cumulative volume 1000ml with water, then use mineral acid or mineral alkali that the solution ph obtained is adjusted to pH7.0 ~ 7.2.
5. cultural method according to claim 2, is characterized in that the preparation method of described fermention medium is as follows:
By 15 ~ 20g starch, 1 ~ 3g corn starch, 1 ~ 5g peptone, 1 ~ 5g sodium-chlor, 1 ~ 5gK
2hPO
43H
2o, 1 ~ 3gNH
4cl and 0.1 ~ 0.5gMgSO
47H
2o is soluble in water, then adds to cumulative volume 1000ml with water, then uses mineral acid or mineral alkali that the solution ph obtained is adjusted to pH7.0 ~ 7.5.
6. the cultural method any one of claim 3 ~ 6 described in claim, is characterized in that described mineral acid is selected from hydrochloric acid or phosphoric acid; Described mineral alkali is selected from sodium hydroxide, potassium hydroxide or ammoniacal liquor.
7. a production method for bacillus amyloliquefaciens LX-J1 preparation, is characterized in that the step of this production method is as follows:
Add in bacillus amyloliquefaciens LX-J1 fermented liquid according to claim 3 with metal ion weight count 0.1 ~ 0.5% copper sulfate, the zinc sulfate of 1 ~ 2%, the manganous sulfate of 0.1 ~ 0.5% and 0.1 ~ 0.6% xanthan gum, obtain described bacillus amyloliquefaciens LX-J1 preparation.
8. production method produces the bacillus amyloliquefaciens LX-J1 preparation obtained according to claim 7, it is characterized in that every milliliter of bacillus amyloliquefaciens LX-J1 preparation contained 10,000,000,000 with last bacillus amyloliquefaciens LX-J1.
9. the purposes of bacillus amyloliquefaciens LX-J1 preparation according to claim 7 in control Rhizoctonia cerealis, dry thread Pyrenomycetes, Pythium ultimum, sclerotium rolfsii and the black hemorrhoid disease of potato.
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