CN1282417C - Microbial bacteriocide, and its preparing method and use - Google Patents
Microbial bacteriocide, and its preparing method and use Download PDFInfo
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- CN1282417C CN1282417C CN 200410040796 CN200410040796A CN1282417C CN 1282417 C CN1282417 C CN 1282417C CN 200410040796 CN200410040796 CN 200410040796 CN 200410040796 A CN200410040796 A CN 200410040796A CN 1282417 C CN1282417 C CN 1282417C
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Abstract
The present invention relates to a microbial bactericide, a preparation method thereof and the application, which belongs to the technical field of a microbial farm chemical. The production strain of the present invention is Brevibacillus laterosporus G4; the strain is preserved at the China type culture substance preservation center on May. 26th 2003, and the preservation number CCTCC No. is M203045. The microbial bactericide of the present invention is prepared by the conventional shake flask fermentation method. The prescription of a solid culture medium comprises 3 g of beef extract, 8 g of peptone, 4 g of sodium chloride, 18 g of agar and 1000 ml of water, and pH is regulated to 7.5. The prescription of a liquid fermentation culture medium comprises 0.2 to 0.4% of beef extract, 10 to 20% of soybean cake powder, 0.5 to 2% fish powder, 0.3 to 0.5% of sodium chloride and water as the rest, and pH is regulated to 7 to 8. When the number of active bacteria is proliferated to 1010/ml, the microbial bactericide for use is prepared. The bactericide of the present invention has good sterilization function for two plant pathogenic bacteria of fusarium and rhizoctonia solani; the present invention which can be used for biologically controlling plant diseases has good application prospects.
Description
Technical field:
The present invention relates to a kind of microbial bactericide and application thereof, the microorganism belonging to genus technical field of pesticide.
Background technology:
China is large agricultural country, and the control of disease pest and weed is the important step in the agricultural production.At present agricultural control of plant disease is still mainly relied on chemical bactericide.But along with scientific technological advance, discover chemical pesticide when preventing and treating disease, more and more serious to the pollution of environment.The while chemical pesticide is the toxicity height often, and killed natural enemies is destroyed bio-diversity, provides condition for more serious damage by disease and insect is rampant, causes the vicious circle on the extermination of disease and insect pest.Human body is produced harm to the residue of pesticide that continuous use causes and the pathogen pesticide resistance is just causing that people pay close attention to greatly.According to FAO (Food and Agriculture Organization of the United Nation) statistics, kind of pathogen has produced pesticide resistance to 1 to several chemical pesticides surplus in the of 100, and analyses and prediction will increase progressively with the quantity in per 5 years about 10% from now on.Agricultural chemicals still is one of inducement of many difficult diseases such as cancer, and Environmental Protection Agency is evaluated 360 kinds of chemical pesticides of home registration, finds that wherein kind more than 70 has carcinogenesis, causes 20,000 routine cancers every year.The chemical control of farming plant pest with preserve the ecological environment, ensure that the contradiction that people ' s health forms more and more causes concern.Microbial pesticide is easy to decompose to the person poultry safety,, is described as " nuisanceless " agricultural chemicals with environmentally compatible.In addition, microbial pesticide also has weak point research cycle, drops into low, as to be easy to industrialization and commercialized development advantage.From eighties of last century since the nineties, microbial pesticide every year has become one of research and development focus in the agricultural biotechnologies industry with 20% speed increase.
Sickle-like bacteria and Rhizoctonia solani Kuhn be two kinds important, the universal phytopathogen.The former almost endangers all plants, causes leaf, stem, the root fusarium wilt of all kinds of plants.The latter causes plant miliary damping-offs such as tobacco, wheat, paddy rice, potato, vegetables, fruit tree.Every plant that has infected above-mentioned two kinds of pathogens, not only output falls sharply, and quality also obviously descends.Fusarium wilt and miliary damping-off that sickle-like bacteria, Rhizoctonia solani Kuhn produce have become critical limitation factor in the agricultural production, and the research and development microbial bactericide has become the important topic in the agricultural sustainable development.
Summary of the invention:
The objective of the invention is by a strain that screens being had the side spore bacillus brevis research of poisoning phytopathogen, exploitation microbial bactericide.
The present invention screens the side spore bacillus brevis Brevibacillus laterosporus G4 that a strain has fine poisoning function to sickle-like bacteria, Rhizoctonia solani Kuhn, and the G4 bacterial strain has been deposited in Chinese typical culture collection center; Address: China. Wuhan. Wuhan University; Preservation date: on May 26th, 2003; The numbering CCTCC NO:M203045 that preservation is registered on the books.
Brevibacillus laterosporus G4 strain morphology of the present invention is characterized as: elongated rod shape, and individuality is bigger, and how the blunt circle in two ends exists with the aggegation form.Rounded, the protuberance of bacterium colony, edge are more neat in the dull and stereotyped training of YPD, mattness, coarse, similar fine powder shape, lawn white.The endospore of cultivating visible only a few more than 36 hours on the YPD medium forms or is discharged into the gemma born of the same parents outside, and at NA or LB medium culture 12-16 hour, the just visible gemma that comes off in a large number, gemma is that ellipse is dugout canoe shape body.
The present invention is achieved in that
The preparation microbial bactericide carries out the bactericide indoor pharmacodynamic test, determines its bactericidal action to sickle-like bacteria and Rhizoctonia solani Kuhn two plant species pathogens.
Preparation microbial bactericide (below be weight percentage):
1, the test tube kind is cultivated
The thalline of G4 is inoculated on the test tube culture medium slant, and culture medium prescription is: beef extract 3 grams; Peptone 8 grams; Sodium chloride 4 grams; Agar 18 grams; Water 1000ml, pH7.5.Cultivated 1-2 days down in 32-38 ℃, obtain the test tube kind.
2, preparation microbial bactericide
Adopt 500ml triangular flask shake-flask culture.Method is first obtaining liq medium, and the liquid culture based formulas is: the 0.2-0.4% beef extract; The 10-20% soybean-cake flour; The 0.5-2% fish meal; Sodium chloride 0.3-0.5%; PH7-8, remainder is a water.The 300ml liquid nutrient medium of in each triangular flask, packing into, 120 ℃ of sterilizations 30 minutes, 1cm is inserted in the cooling back
2Big test tube kind, under 37 ℃, the 120rpm/min shaking table was cultivated 2 days, bred to the top when viable count, reached 10
10During/ml, promptly become operational microbial bactericide.
The microbial bactericide indoor pharmacodynamic test:
1, preparation test with medicament
By aforementioned test tube kind cultural method culture test tube kind, by aforementioned preparation microbial disinfection agent method preparation test with medicament.
2, preparation contrast with medicament
Contrast 1: after preparing microbial bactericide by aforementioned preparation bactericide method, filtering out thalline with biofilter, is contrast with the zymotic fluid of mycetome not.
Contrast 2: aforementioned 2 preparation bactericide method preparation contrast with medicament, but do not insert the G4 bacterial strain in the liquid nutrient medium, be contrast with nonvaccinated zymotic fluid.
Contrast 3: in contrast with clear water.
2, pathogen is used in the preparation test
Reaping hook germ and miliary damping-off germ are inoculated into respectively on the test tube culture medium slant, the prescription of medium is PDA medium commonly used, method is to be cut into small pieces after getting potato 200 gram peelings, adding water 1500ml boiled 20 minutes, obtain 1000ml filtrate after the filtration, add glucose 20 grams then, agar 18 grams, pH nature.Cultivated 2-3 days down in 25 ℃, after waiting mycelia to cover with and producing spore, wash stand-by with sterile water.
3, test method
Preparation mixes the bacterium flat board: after the culture dish sterilization with diameter 9ml, pour 20ml45 ℃ PDA medium and 0.1ml pathogen liquid into, fully make reaping hook germ, the mixed bacterium flat board of miliary damping-off germ behind the mixing.
The test of pesticide effectiveness adopts conventional Oxford cup bacteriostatic experiment method, promptly on each mixed bacterium flat board, place the Oxford cup, in the cup of Oxford, add test with medicament and the contrast with medicament of 0.1ml, cultivated 72 hours down in 25 ℃, measure antibacterial circle diameter (deduction Oxford cup diameter), repeat three samples.
4, result of the test
Table 1 microbial bactericide is to reaping hook germ indoor harmacological effect
| Pathogen | Test with medicament antibacterial circle diameter | Contrast 1 antibacterial circle diameter | Contrast 2 antibacterial circle diameters | Contrast 3 antibacterial circle diameters |
| Sickle-like bacteria 1 | 2.1cm | 0 | 0 | 0 |
| Sickle-like bacteria 2 | 1.9cm | 0 | 0 | 0 |
| Sickle-like bacteria 3 | 2.3cm | 0 | 0 | 0 |
| On average | 2.1cm | 0 | 0 | 0 |
Table 2 microbial bactericide is to miliary damping-off germ indoor harmacological effect
| Pathogen | Test with medicament antibacterial circle diameter | Contrast 1 antibacterial circle diameter | Contrast 2 antibacterial circle diameters | Contrast 3 antibacterial circle diameters |
| Rhizoctonia solani Kuhn 1 | 3.9cm | 0 | 0 | 0 |
| Rhizoctonia solani Kuhn 2 | 3.8cm | 0 | 0 | 0 |
| Rhizoctonia solani Kuhn 3 | 3.5cm | 0 | 0 | 0 |
| On average | 3.73cm | 0 | 0 | 0 |
The result shows that microbial bactericide of the present invention has bactericidal action preferably to sickle-like bacteria, Rhizoctonia solani Kuhn two plant species pathogens, and average antibacterial circle diameter reaches 2.1 and 3.73cm respectively, and miliary damping-off germ effect is better than reaping hook germ effect.But its average inhibition zone all more than 2cm, has shown application promise in clinical practice.
Contrasting 1 the bacteriostasis that has no, show that bactericidal action is produced by the viable bacteria body, is not conventional bacterial metabolism product, and emphasis is considered increase viable bacteria body burden in industrialization is produced.
Embodiment:
Below be embodiments of the invention, but content of the present invention is not limited thereto.
Embodiment one:
Brevibacillus laterosporus G4 thalline is inoculated on the test tube agar medium inclined-plane, and culture medium prescription is: beef extract 3 grams; Peptone 8 grams; Sodium chloride 4 grams; Agar 18 grams; Water 1000ml, pH7.5; Cultivated 2 days down in 32 ℃, obtain the test tube kind;
With 500ml triangular flask enlarged culture, the liquid culture based formulas is: 0.3% beef extract; 15% soybean-cake flour; 1% fish meal; Sodium chloride 0.4%; PH7.5; Remainder is a water.The 300ml liquid nutrient medium of in each triangular flask, packing into, 120 ℃ of sterilizations 30 minutes, the test tube kind of 1 square of ml size is inserted in the cooling back, and under 37 ℃, 120rpm/min cultivated 2 days, bred to the top when viable count, reached 10
10During/ml, promptly become operational microbial bactericide.
Embodiment two:
Substantially with embodiment one, difference is: the liquid culture based formulas is: 0.2% beef extract, 10% soybean-cake flour, 0.5% fish meal, sodium chloride 0.3%, pH7, remainder are water; It is that 35 ℃, incubation time are 1.5 days that the test tube kind is incubated at temperature.
Embodiment three:
Substantially with embodiment one, difference is: the liquid culture based formulas is: 0.4% beef extract, 20% soybean-cake flour, 2% fish meal, sodium chloride 0.5%, pH8, remainder are water; It is that 38 ℃, incubation time are 1 day that the test tube kind is incubated at temperature.
Claims (2)
1, a kind of microbial bactericide, prepare through conventional method by producing bacterial strain, the production bacterial strain that it is characterized in that this bactericide is side spore bacillus brevis Brevibacillus laterosporus G4, this bacterial strain has been deposited in Chinese typical culture collection center, preserving number CCTCC NO:M203045 on May 26th, 2003.
2, the application of the described microbial bactericide of claim 1 is characterized in that this bactericide to sickle-like bacteria and Rhizoctonia solani Kuhn two plant species pathogens are had bactericidal action, can be used for biocontrol of plant disease.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410040796 CN1282417C (en) | 2004-09-30 | 2004-09-30 | Microbial bacteriocide, and its preparing method and use |
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| Application Number | Priority Date | Filing Date | Title |
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| CN 200410040796 CN1282417C (en) | 2004-09-30 | 2004-09-30 | Microbial bacteriocide, and its preparing method and use |
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| CN1640266A CN1640266A (en) | 2005-07-20 |
| CN1282417C true CN1282417C (en) | 2006-11-01 |
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| CN 200410040796 Expired - Fee Related CN1282417C (en) | 2004-09-30 | 2004-09-30 | Microbial bacteriocide, and its preparing method and use |
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102086398A (en) * | 2010-12-31 | 2011-06-08 | 东莞市保得生物工程有限公司 | Soil conditioner |
| CN103283944A (en) * | 2013-06-09 | 2013-09-11 | 广州格拉姆生物科技有限公司 | Solid fermentation production method of brevibacillus laterosporus WY9701 preparation |
| CN106748259A (en) * | 2017-01-17 | 2017-05-31 | 四川鹤岛农业科技有限公司 | A kind of retain water and nutrients composite microbic bacterial fertilizer and application |
| CN107151641B (en) * | 2017-07-04 | 2020-11-27 | 沈阳农业大学 | Brevibacillus laterosporus for inhibiting rhizoctonia solani and application thereof |
| CN107267425A (en) * | 2017-08-03 | 2017-10-20 | 北京泰克美高新技术有限公司 | A kind of method of preparation and use of fresh-keeping use active microbial inoculum |
| CN111269865B (en) * | 2020-04-01 | 2020-12-01 | 北京工商大学 | Brevibacillus laterosporus strain S62-9 and application thereof |
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