CN105441488A - Antibacterial crude extract generated by bacillus M29 with broad-spectrum soil-borne pathogenic bacterium antagonistic function and application of antibacterial crude extract - Google Patents
Antibacterial crude extract generated by bacillus M29 with broad-spectrum soil-borne pathogenic bacterium antagonistic function and application of antibacterial crude extract Download PDFInfo
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Abstract
The invention discloses an antibacterial crude extract generated by bacillus M29 with the broad-spectrum soil-borne pathogenic bacterium antagonistic function and application of the antibacterial crude extract. The bacillus M29 is preserved in China General microbiological culture collection center of China Management Committee for Culture Collection of Microorganisms, the preservation serial number is CGMCC NO.11557, and the preservation date is October 29, 2015. The antibacterial crude extract extracted through an acidic precipitation method after fermentation liquor of the strain is subjected to centrifugal sterilization can still effectively antagonize grey mould, trichoderma, aspergillus fumigatus, fusarium oxysporum, fusarium oxysporum vasinfectum, muskmelon fusarium wilt, strawberry root rot fungi, peach canker bacteria, verticillium dahilae, rhizoctonia solani kuhn and other pathogenic fungi. The antibacterial crude extract has the advantages of being easy to preserve, capable of being massively produced, good in prevention effect, good in environment safety and the like, and has good commercial application prospects.
Description
Technical field
The present invention relates to a kind of microorganism, relate in particular to antibacterial crude extract and the application thereof of the genus bacillus M29 generation with wide spectrum antagonism soil-borne pathogen function.
Background technology
The biological control of Plant diseases refers to and reduces pathogen quantity by one or more biologies except people or weaken the pathogenic vigor of cause of disease, thus reduces the generation of disease caused by pathogen.There is in wormcast abundant beneficial microorganism, can effective antagonism soil-borne disease fungal pathogens.It is reported that antagonistic microbe is by producing the mechanism such as system resistant to suppress pathogenic bacteria with the antibiosis of pathogenic bacteria, Competition, hyperparasitism and inducing plant, reaches the object of controlling plant diseases.In addition, antagonistic microbe is environmentally safe, environmental pollution and the murder by poisoning to people and animals can not be caused as chemical pesticide, antagonistic microbe not easily makes pathogenic bacteria produce resistance simultaneously, some also has the effect of Promoting plant growth, and actual production technique is simple, therefore utilizes the research of antagonistic microbe controlling disease to be just more and more subject to the attention of domestic and international scientific research institution.At present, people have been separated to the multiple antagonistic microbe various different Plant diseases being had to prevention effect in various degree, and wherein some is own through entering practical stage, creates suitable social benefit and environmental benefit.
Summary of the invention
The object of the invention is the above-mentioned deficiency for prior art, the antibacterial crude extract of the generation of the genus bacillus M29 with wide spectrum antagonism soil-borne pathogen function is provided.
Another object of the present invention is to provide the application of the antibacterial crude extract that this genus bacillus produces.
Object of the present invention realizes by following technical scheme:
One strain has the genus bacillus M29 of wide spectrum antagonism soil-borne pathogen function, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCCNO.11557, and preservation date is on October 29th, 2015.
Described genus bacillus M29 bacterium colony is on LB flat board, and Initial stage of culture bacterium colony is full transparent, accumulates a large amount of mucus, is attached on substratum securely, and late stage of culture is decomposite leaf shape, and edge is hair-like, and after cultivation 3d, bacterium colony is the massif shape that fold has been built.Can produce ellipticity gemma raw closely, sporangiocyst slightly expands, and cellular form and arrangement are shaft-like, and Dan Sheng is without pod membrane, movable.
The physio-biochemical characteristics of described genus bacillus M29 are: Gram-positive, strictly aerobic, chemoheterotrophy, M.R negative, and VP tests the positive, and Starch Hydrolysis is positive, and gelatin hydrolysis is negative, and Citrate trianion utilizes positive, and nitrate reduction is positive.
The major nitrogen source used when genus bacillus M29 of the present invention cultivates includes but not limited to peptone, yeast powder, saltpetre, ammonium nitrate, ammonium sulfate, urea, L-Ala; The primary carbon source used includes but not limited to sucrose, wood sugar, N.F,USP MANNITOL, maltose, lactose, fructose, glucose; The inorganic component used includes but not limited to Repone K, sodium-chlor, SODIUM PHOSPHATE, MONOBASIC, dipotassium hydrogen phosphate, tricalcium phosphate, Calcium dichloride dihydrate, bitter salt, seven water and ferrous sulfate.Genus bacillus M29 fermentation of the present invention can at 25 ~ 37 DEG C, and pH4 ~ 10, sodium chloride concentration carries out under the environment of 1%-10%.
The antibacterial crude extract that genus bacillus M29 of the present invention produces, prepare by the following method: genus bacillus M29 of the present invention is fermented, fermented liquid is centrifugal, gained supernatant liquor is degerming with sterilized 0.22 μm of hole membrane filtration, is considered as bacteria-free filtrate through slat chain conveyor without bacterium colony formation; After cultivation terminates, centrifuging and taking supernatant liquor, adds 6molLHCL and regulates pH to 2.0 in supernatant liquor, 4 DEG C are spent the night, centrifugal, collecting precipitation, by the washed several times with water of pH2.0, methyl alcohol extracting is added 2 times after precipitation drying, merge extract, rotary evaporation, obtains pale yellow powder, be dissolved in the phosphoric acid buffer of pH6.8, then namely obtain antibacterial crude extract with after the membrane filtration of 0.22 μm.
The application of antibacterial crude extract of the present invention in the biological prevention and control agent preparing antagonism grey mold, mould, the Aspergillus fumigatus of wood, cucumber Fusarium oxysporum, cotton wilt fusarium, muskmelon Fusarium oxysporum, strawberry root-rot bacterium, peach ulcer bacterium, verticillium dahliae and/or Rhizoctonia solani.
Beneficial effect:
The invention provides a strain to can the effective multiple pathogenic bacteria such as antagonism grey mold, mould, the Aspergillus fumigatus of wood, cucumber Fusarium oxysporum, cotton wilt fusarium, muskmelon Fusarium oxysporum, strawberry root-rot bacterium, peach ulcer bacterium, verticillium dahliae, Rhizoctonia solani, meanwhile, the antibacterial crude extract extracted through acid precipitation method after the fermented liquid bactofugation of this bacterial strain still all can the effective pathogenic fungi such as antagonism grey mold, wooden mould, Aspergillus fumigatus, cucumber Fusarium oxysporum, cotton wilt fusarium, muskmelon Fusarium oxysporum, strawberry root-rot bacterium, peach ulcer bacterium, verticillium dahliae, Rhizoctonia solani; The advantages such as have easy preservation, can produce in a large number, preventive effect is good, and environmental safety is good, have good commercial application prospect.
Accompanying drawing explanation
Fig. 1 M29 microscope ocular 10 ×, oily mirror 100 × under form
Fig. 2 M29 bacterial strain is to the antagonistic effect of grey mold.
The antagonistic effect that Fig. 3 M29 bacterial strain is withered to cucumber.
Fig. 4 M29 bacterial strain is to the antagonistic effect of strawberry root-rot.
The antagonistic effect that Fig. 5 M29 bacterial strain is withered to cotton.
Fig. 6 M29 bacterial strain is to the antagonistic effect of peach ulcer.
Fig. 7 M29 bacterial strain is to beautiful greatly antagonistic effect of taking turns branch.
The antagonistic effect that Fig. 8 M29 bacterial strain is mould to wood.
The antagonistic effect that Fig. 9 M29 bacterial strain is withered to muskmelon.
Figure 10 M29 bacterial strain is to the antagonistic effect of Aspergillus fumigatus.
Figure 11 M29 bacterial strain is to the antagonistic effect of rice banded sclerotial blight.
Figure 12 M29 bacterial strain produces antimicrobial substance to the antagonistic effect of grey mold.
Figure 13 M29 bacterial strain produces the antagonistic effect that antimicrobial substance is withered to cucumber.
Figure 14 M29 bacterial strain produces antimicrobial substance to the antagonistic effect of strawberry root-rot.
Figure 15 M29 bacterial strain produces the antagonistic effect that antimicrobial substance is withered to cotton.
Figure 16 M29 bacterial strain produces antimicrobial substance to the antagonistic effect of peach ulcer.
Figure 17 M29 bacterial strain produces antimicrobial substance to beautiful greatly antagonistic effect of taking turns branch.
Figure 18 M29 bacterial strain produces the antimicrobial substance antagonistic effect mould to wood.
Figure 19 M29 bacterial strain produces the antagonistic effect that antimicrobial substance is withered to muskmelon.
Figure 20 M29 bacterial strain produces antimicrobial substance to the antagonistic effect of Aspergillus fumigatus.
Figure 21 M29 bacterial strain produces antimicrobial substance to the antagonistic effect of rice banded sclerotial blight.
Biomaterial preservation information
M29, Classification And Nomenclature is Bacillus subtilis, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, deposit number is CGMCCNO.11557, and preservation date is on October 29th, 2015.
Embodiment
Embodiment 1
First following three kinds of substratum are prepared.
LB solid medium: peptone 10g, yeast powder 5g, sodium-chlor 10g, agar 30g, distilled water 1000ml, pH7.0-7.2,121 DEG C of sterilizings, 20min.
LB liquid nutrient medium: do not add agar, other condition is the same.
PDA substratum: glucose 20.0g, agar 20.0g, potato powder 6.0g, distilled water 1000ml, pH5.4-5.8,115 DEG C of sterilizings, 30mine.
YPD substratum: glucose 20g, peptone 20g, yeast powder 10g, agar 30g, distilled water 1000ml, pH7.0-7.2,115 DEG C of sterilizings, 30mine.
Wormcast is taken the triangular flask that l0g is placed in the 250ml filling 100ml aqua sterilisa, in shaking table, 30 DEG C, 150rmin
-1vibration 20min, leaves standstill 10min, obtains suspension.Containing several bacterium in this suspension, be applied to LB substratum, be inverted by flat board after adopting the dilution of gradient dilution method, in 30 DEG C, after cultivating 24h in thermostat container, the single bacterium colony of the dissimilar typical case of picking, after dull and stereotyped purifying, 4 DEG C to be kept at LB inclined-plane stand-by.
Below again by the antagonistic action of dull and stereotyped this bacterium of face-off experimental verification.
Primary dcreening operation: respectively that wood is mould, grey mold, Aspergillus fumigatus, verticillium dahliae, cotton wilt fusarium, muskmelon Fusarium oxysporum, peach ulcer bacterium, careless mould root-rot bacterium is dull and stereotyped central in PDA with transferring after punch tool punching, cucumber Fusarium oxysporum, transfer after the punching of Rhizoctonia solani punch tool in YPD substratum central authorities, then the bacterium picking list bacterium colony point after separation and purification is connected on the corner of fungi, be placed in 30 DEG C of incubators and cultivate, grey mold is placed in 25 DEG C of incubators, cultivate 3-5d, select effective bacterium according to fungistatic effect.
Multiple sieve: the effective bacterium obtained by primary dcreening operation is repeated according to above-mentioned experimental technique, and having filtered out a strain all has antagonistic action and the bacterial strain of successful to ten kinds of fungal pathogens, called after M29 (Fig. 2 ~ Figure 11).
As shown in Figure 1, this bacterial strain oily mirror 100 ×, eyepiece 10 × microscopic conditions under morphological specificity.
By the bacterial strain that aforesaid method screening and separating goes out, through the order-checking of Nanjing Si Pujin specialty order-checking company, according to the sequencing result of 16SrDNA, analyze in http://www.ncbi.nlm.nih.gov online query, Blast software is utilized to carry out tetraploid rice with other 16SrDNA sequence in GenBank, determine that it is genus bacillus, be then defined as subtilis by Biolog result and physiological and biochemical property.This bacterial strain is delivered the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, deposit number is CGMCCNO.11557, and preservation date is on October 29th, 2015.
This bacterium is Gram-positive, strictly aerobic, chemoheterotrophy, M.R negative, and VP tests the positive, the Starch Hydrolysis positive, and gelatin hydrolysis is negative, and Citrate trianion utilizes positive, and nitrate reduction is positive, has wide spectrum and obvious germ resistance.
Embodiment 2
Aerobic is tested
Sterilized LB substratum is poured in 3 sterilized test tubes, and greatly about 2/3 place, on aseptic operating platform, with the described bacterium of inoculating needle picking slant culture, percutaneous puncture-inoculation (must be punctured at the bottom of pipe) in above-mentioned substratum.30 DEG C of cultivations, respectively 3 days to 7 days observationss.Be aerobic bacteria agar column surface-borne person, as being anerobe or facultative anaerobe along the raw elder of puncture line.Test-results shows, and bacterium colony is along agar column surface growth, and puncture line is interior without colony growth, for strictly aerobic.
Catalatic mensuration
Clean slide drips 1 3%H
2o
2, get LB slant culture 1 ring of 18 ~ 24h, at H
2o
2in smear, if there is bubble to produce, be positive, otherwise be feminine gender.Test-results is that catalase is positive.
Methyl red test (M.R test)
A. substratum and reagent: peptone 5g, glucose 5g, sodium-chlor 5g, distilled water 1000mL, regulate pH7.0 ~ 7.2, packing test tube, often pipe fills 4 ~ 5mL, 121 DEG C of sterilizing 20min.Reagent: methyl red 0.1g, 95% alcohol 300mL, distilled water 200mL.
B. spawn culture and result observe inoculating strain in above-mentioned nutrient solution, cultivate l ~ 2 day for 30 DEG C.In nutrient solution, add several methyl red reagent, as nutrient solution presents redness, for methyl red is positive, yellow is negative (methyl red color change interval 4.4 redness ~ 6.0 is yellow).
Test-results is that M.R is negative.
Second phthalein carbinol methine test (VP test)
A. the same methyl red test of culture medium culturing base.
B. spawn culture and result are observed inoculation and are cultivated same methyl red test.When doing VP test, get nutrient solution (about 2mL) and mix with the 40%NaOH phase of equivalent, add a small amount of creatine, after the 2 ~ 5min that fully vibrates, as nutrient solution occurs red, be the VP positive.
Test-results is that VP is positive.
Starch Hydrolysis is tested
A. substratum and reagent add the Zulkovsky starch of 0.2% in meat soup peptone agar, and packing triangular flask, 121 DEG C of sterilizing 20min are for subsequent use.Road Ge Shi iodine liquid: crystalline flake of iodine 1g, potassiumiodide 2g, first uses a small amount of (3 ~ 5mL) distilled water to dissolve potassiumiodide, now adds the crystalline flake of iodine, after iodine dissolves completely, be diluted with water to 300mL.
B. spawn culture and result are observed and are got bacterial classification point and be connected on flat board, cultivate 2 ~ 4 days for 30 DEG C, after forming bacterium colony, flat board drips road Ge Shi iodine liquid, to be paved with periphery of bacterial colonies for degree, dull and stereotyped in blue, and periphery of bacterial colonies is irised out existing if any water white transparency, illustrate that starch is hydrolyzed.The size of the size general remark hydrolyzed starch ability of transparent circle.
Test-results is that Starch Hydrolysis is positive.
Gelatin hydrolysis is tested
A. substratum and reagent peptone 5g, gelatin 120g, distilled water 1000mL.Regulate pH7.2 ~ 7.4, packing test tube, substratum height is about 4 ~ 5cm, 121 DEG C of sterilizing 20min.
B. spawn culture and result observation puncture method are seeded in test tube central authorities.Cultivate one month in 30 DEG C of incubators, observe gelatin and whether liquefy.
Test-results is that gelatin hydrolysis is negative.
Nitrate reduction test
A. substratum and reagent peptone 10g, KNO
31g, distilled water 1000mL, pH7.0 ~ 7.4.Ge Lisishi (Gries) reagent: A liquid: Sulphanilic Acid 0.5g, dilute acetic acid (about 10%) 150mL; B liquid: Cai's amine 0.1g, distilled water 20mL, dilute acetic acid (about 10%) 150mL.Pentanoic reagent: pentanoic 0.5g is dissolved in the l00mL vitriol oil, uses 20mL distilled water diluting.
B. spawn culture and result are observed and are inoculated in nitrate liquid nutrient medium by test organisms, cultivate 1,3,5 day for 30 DEG C.In white porcelain dish aperture, pour a little nutrient solution into, then drip 1 reagent A and B liquid wherein respectively, when nutrient solution become pink, rose, orange or brown etc. time, indicate that nitrite exists, be that nitrate reduction is positive, otherwise be feminine gender.
Test-results is that nitrate reduction is positive.
The utilization of Citrate trianion
A. substratum and reagents citric acid sodium 2g, NaCl5g, MgSO
47H
2o0.2g, (NH
4)
2hPO
41g, 1% Australia thymol blue aqueous solution 10mL, agar 20g, distilled water 1000mL, pH6.8-7.0,121 DEG C of sterilizing 20min.
B. spawn culture and result are observed and are got children's strain inoculation in age on inclined-plane, and cultivate 3-7 days for 30 DEG C, substratum is positive reaction in alkalescence (blueness) person, and constant person is then negative.
The test-results that Citrate trianion utilizes is the positive.
Embodiment 3
Prepare substratum:
Landy: glucose 20g, L mono-L-glutamic acid 5g, KH
2pO
4; 1.5g, MgSO
47H
2o0.5g, KCI0.5g, yeast powder 1g, MnSO; 5mg, CuSO
45H
2o0.16mg, FeSO47H
2o0.15mg, L mono-phenylalanine 2mg, distilled water IL, pH7.0
The M29 seed liquor of bacterial strain is cultivated:
By the M29 strain transfer of preservation on LB flat board, cultivate 24h for 37 DEG C; Get a ring activated spawn list colony inoculation in the 250ml triangular flask containing 100mlLB liquid nutrient medium with transfering loop, 37 DEG C, 170r/min, cultivate 24h as seed liquor.
The preparation of bacteria-free filtrate and antimicrobial substance crude extract:
The preparation of bacteria-free filtrate: seed liquor is inoculated into 100mlLandy substratum by the inoculum size with 5%, after certain CMC model, fermented liquid 4 DEG C, 12000r/min, centrifugal 20min, degerming with sterilized 0.22 μm of hole membrane filtration, be considered as bacteria-free filtrate through slat chain conveyor without bacterium colony formation.
The preparation of antimicrobial substance crude extract: the extraction of antimicrobial substance adopts acid precipitation method.After cultivation terminates, the centrifugal l0min of 12000rmin', gets supernatant liquor, and in supernatant liquor, add 6molLHCL regulate pH to 2.0,4 DEG C are spent the night.8000rmin is centrifugal 10min again, collecting precipitation, by the washed several times with water of pH2.0, methyl alcohol extracting is added 2 times after precipitation drying, merge extract, rotary evaporation, obtains pale yellow powder, be dissolved in the phosphoric acid buffer (PBs) of pH6.8, then namely obtain aseptic crude extract with after the membrane filtration of 0.22 μm.
Embodiment 4
Antimicrobial substance compliance test result:
Wood is mould, grey mold, Aspergillus fumigatus, verticillium dahliae, cotton wilt fusarium, muskmelon Fusarium oxysporum, peach ulcer bacterium, the mould root-rot bacterium of grass is with transferring in the dull and stereotyped central authorities of PDA after punch tool punching, cucumber Fusarium oxysporum, transfers after the punching of Rhizoctonia solani punch tool in YPD substratum central authorities, then after aseptic filter paper sheet being soaked in the phosphoric acid buffer of pH6.8 being dissolved with antibacterial crude extract, be placed on pathogenic bacteria corner, be placed on 30 DEG C of incubators and cultivate, grey mold is placed in 25 DEG C of incubators and cultivates.Observing effect after 2-3 days, the visible accompanying drawing 12 ~ Figure 21 of effect.
The above is only basic embodiment of the present invention; be noted that for those skilled in the art; under the premise without departing from the principles of the invention, can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (2)
1. the antibacterial crude extract of genus bacillus M29 generation, it is characterized in that described genus bacillus M29, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCCNO.11557, and preservation date is on October 29th, 2015; Described antibacterial crude extract prepares by the following method: fermented by described genus bacillus M29, fermented liquid is centrifugal, and gained supernatant liquor is degerming with sterilized 0.22 μm of hole membrane filtration, is considered as bacteria-free filtrate through slat chain conveyor without bacterium colony formation; After cultivation terminates, centrifuging and taking supernatant liquor, adds 6mRlLHCL and regulates pH to 2.0 in supernatant liquor, 4 DEG C are spent the night, centrifugal, collecting precipitation, by the washed several times with water of pH2.0, methyl alcohol extracting is added 2 times after precipitation drying, merge extract, rotary evaporation, obtains pale yellow powder, be dissolved in the phosphoric acid buffer of pH6.8, then namely obtain antibacterial crude extract with after the membrane filtration of 0.22 μm.
2. the application of antibacterial crude extract according to claim 1 in the biological prevention and control agent preparing antagonism grey mold, mould, the Aspergillus fumigatus of wood, cucumber Fusarium oxysporum, cotton wilt fusarium, muskmelon Fusarium oxysporum, strawberry root-rot bacterium, peach ulcer bacterium, verticillium dahliae and/or Rhizoctonia solani.
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