CN102396539A - Earthworm antimicrobial peptide biological pesticide and application - Google Patents
Earthworm antimicrobial peptide biological pesticide and application Download PDFInfo
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- CN102396539A CN102396539A CN2010102785658A CN201010278565A CN102396539A CN 102396539 A CN102396539 A CN 102396539A CN 2010102785658 A CN2010102785658 A CN 2010102785658A CN 201010278565 A CN201010278565 A CN 201010278565A CN 102396539 A CN102396539 A CN 102396539A
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Abstract
The invention provides an earthworm antimicrobial peptide biological pesticide and application and belongs to the field of biochemistry. A technical scheme is as below: extracting earthworm antimicrobial peptide from earthworm body cavity and tissue homogenate by a phosphate buffer; centrifuging a leaching liquor; carrying out ultrafiltration; and concentrating to prepare the earthworm antimicrobial peptide biological pesticide. The biological pesticide of the invention can be effectively applied to control of insect diseases of paddy rice, fruit and vegetable; and the production technology is simple and without generating pollution. The biological pesticide of the invention belongs to a green environment friendly product without pollution, and is especially suitable for production of nuisance-free organic agricultural products.
Description
Technical field
The present invention relates to a kind of earthworm antibacterial peptide biopesticide and the application in crop pest control.Be specifically related to a kind of preparation of earthworm antibacterial peptide biopesticide and the application of prevention and elimination of disease and pests in paddy rice and fruits and vegetables production.Belong to biological chemical field.
Background technology
The long-term cultivated area of staple food crop paddy rice of China surpasses 2,800 ten thousand hectares, and the vegetable melon and fruit cultivated area is above 1800 hectares. and wherein extermination of disease and insect pest area is about 6,700,000,000 mu.It is annual because the generation of damage by disease and insect all can cause these economic crops larger area underproduction and quality to reduce.
Present main Prevention Technique still relies on chemical pesticide.A large amount of chemical pesticides that use have made China become one of the most serious country of World chemical agricultural chemicals mud, have caused thus that poisonous residue of pesticide detected level generally exceeds standard in agricultural products in China and the food.And then harm people's food security and health.In international trade, the outlet of annual agricultural products in China answer agricultural chemicals to exceed standard and blocked by foreign country's " green barrier " causing with a toll of more than 70 hundred million U.S.As.
The extermination of disease and insect pest had crucial meaning during the research and development of biopesticide were produced for China's grain, vegetables with production; Be to solve current agricultural product and the residual important channel that exceeds standard of the poisonous agricultural chemicals of food, especially in producing the organic farm products process, rise and do key effect.
Antibacterial peptide (Antimicrobial pepertides) is the small molecule polypeptide that has antibacterial activity in the biology.Antibacterial peptide has has a broad antifungal spectrum, and effect is strong, and bad reaction is little, and safe range is big, non-environmental-pollution, the characteristics that do not develop immunity to drugs.Antibacterial peptide extensively is present in many biologies.Earthworm lives in and is full of various microorganisms, in addition in the environment of a large amount of pathogenic microorganismss, the immunologic mechanism of the opposing adventive invasive ability that forms through long-term evolution, synthetic is its important immunologic function with secreting antibacterial peptide.Recent study is found in juice of earthworm and tissue homogenate, to contain multiple antibacterial peptide.Early studying the earthworm antibacterial peptide is (1985) such as Vaillier, and they extract 3 kinds of antibacterial peptides with very strong antibacterial activity from coelomic fluid of earthworm; Alexandra Milochau (1997) has been separated to two kinds of antibacterial peptides from the earthworm body; Chao etc. (1997) obtain a kind of antibacterial polypeptide Lumbricus I from earthworm Lumbricus rubellus, experiment in vitro shows that it has broad-spectrum antibacterial activity.Sun Zhenjun (1995) research is separated to a kind of antibacterial peptide (four decapeptides) Zhang Xichun (2002) etc. and is separated to two kinds of antibacterial peptides, all has tangible antibacterial effect.In addition, Jia Jiuman etc. (2007), Zheng Jinhui etc. (2008) show to purification of antibacterial peptides from earthworm and active result of study that earthworm antibacterial peptide extract is external has a tangible antibacterial activity.Therefore visible, the earthworm antibacterial peptide can be used as the plain resource of a kind of desirable biological antibiotic and researches and develops.The patent of invention of Sun Zhenjun (00136778.1) discloses a kind of preparation method of earthworm peptide antibacterial peptide, and the patent of invention of Sun Zhenjun (200310115583.4) discloses a kind of earthworm active peptide antiseptic feed raw material and production method.The earthworm antibacterial peptide has been able to use aspect feed.But method can also further be improved.So far, data-searching is not seen earthworm antibacterial peptide agricultural chemicals as yet and is used to spray paddy rice and the report of vegetable melon and fruit cauline leaf prevention and elimination of disease and pests.
Summary of the invention
To the biopesticide that lacks in the prior art with earthworm antibacterial peptide control paddy rice and gourd, fruit and vegetable damage by disease and insect, the present invention aims to provide a kind of earthworm antibacterial peptide biopesticide and is used to spray paddy rice and gourd, fruit and vegetable cauline leaf or seed soaking prevention and elimination of disease and pests.
The present invention realizes through following technical scheme:
(1) a kind of preparation method of earthworm antibacterial peptide biopesticide may further comprise the steps: get earthworm alive and in water, soak 2-4h, wash foreign material after, use distilled water flushing again 3 times, put into an earthworm and put into a tank that has positive and negative electrode and link to each other with controller; Add the phosphate buffer (pH5.8～6.0) of 2 times of volumes, energized is under voltage 15～20V, to earthworm electro photoluminescence 0.5～1.5min; Earthworm is taken out, earthworm is rubbed, add 6-8 times of volume phosphate buffer (pH5.8～6.0), lixiviate 2-4h with tissue refiner; Be heated to 80 ℃ again, insulation 15min, after the cooling, centrifugation; It is subsequent use to obtain centrifugate, and buffer solution and above-mentioned centrifugate that other takes out in the tank merge, with the membrane filtration of 1 μ m; Collect filtrating, use molecular cut off to carry out ultrafiltration then, collect ultrafiltrate as the milipore filter of 50kD; Ultrafiltrate is carried out ultrafiltration with the 1kD milipore filter again, collect trapped fluid, the trapped fluid of gained is the antibacterial peptide biopesticide.The earthworm raw material that the present invention is used to produce antibacterial peptide is that the compatriot likes to win earthworm.
(2), a kind of method for using of earthworm antibacterial peptide biopesticide:
With 200～400 times of uses of earthworm antibacterial peptide biopesticide dilute with water, when being used for paddy rice, carrying out cauline leaf in seedling stage, vegetative period, flowering stage, pustulation period respectively and spray; When being used for gourd, fruit and vegetable, carrying out cauline leaf in seedling stage, vegetative period, flowering stage, fruit expanding period respectively and spray; The solution of 200～400 times of dilutions is adopted in paddy rice, gourd, fruit and vegetable seed soaking.
Beneficial effect of the present invention
Useful effect of the present invention is powdery mildew, gray mold, anthracnose and the budworm disease that this biopesticide can effectively be used to control rice blast, banded sclerotial blight, rice leaf roller, rice fulgorid, gourd, fruit and vegetable, can also promote plant growth and the quality that improves crop.The production technology of this agricultural chemicals is simpler, and production process does not produce pollution, and this agricultural chemicals Nantural non-toxic belongs to the friendly product of environmentally safe green environment.Especially be suitable for producing nuisanceless organic farm products.
Embodiment
Through embodiment the present invention is described in further detail below,
Embodiment 1
The preparation of earthworm antibacterial peptide biopesticide: get earthworm 1000g alive and in water, soak 2h, wash foreign material after, use distilled water flushing again 3 times, put into the tank that positive and negative electrode is installed and links to each other with controller; Add 2000mL phosphate buffer (pH5.8), the liquid level of buffer solution is higher than electrode, and energized is under the 20V at voltage; To earthworm electro photoluminescence 0.5min, the earthworm of irriate is discharged juice and gets into phosphate buffer, and earthworm is taken out, and rubs with tissue refiner; Add 8000mL phosphate buffer (pH5.8), light and slow stirring and leaching 3h is heated to 80 ℃ again, insulation 15min; After the cooling, with 4000r/m, centrifugal 15min, it is subsequent use to obtain centrifugate; Buffer solution and above-mentioned centrifugate that other takes out in the tank merge, and with the membrane filtration of 1 μ m, collect filtrating, use molecular cut off to carry out ultrafiltration as the milipore filter of 50kD then; Collect ultrafiltrate, use the 1kD milipore filter to carry out ultrafiltration to trapped fluid again ultrafiltrate and be 250mL, the trapped fluid of gained is the antibacterial peptide biopesticide.
The application process of earthworm antibacterial peptide biopesticide: at rice growing season, get 200 times of the said goods 200mL thin ups after, carry out stems and leaves of rice and spray.
Embodiment 2
The preparation of earthworm antibacterial peptide biopesticide: get earthworm 1000g alive and in water, soak 3h, wash foreign material after, use distilled water flushing again 3 times, put into the tank that positive and negative electrode is installed and links to each other with controller; Add 2000mL phosphate buffer (pH5.8), the liquid level of buffer solution is higher than electrode, and energized is under the 15V at voltage; To earthworm electro photoluminescence 1.0min, the earthworm of irriate is discharged juice and gets into phosphate buffer, and earthworm is taken out, and rubs with tissue refiner; Add 7000mL phosphate buffer (pH5.8), light and slow stirring and leaching 4h is heated to 80 ℃ again, insulation 15min; After the cooling, with 4000r/m, centrifugal 15min, it is subsequent use to obtain centrifugate; Buffer solution and above-mentioned centrifugate that other takes out in the tank merge, and with the membrane filtration of 1 μ m, collect filtrating, use molecular cut off to carry out ultrafiltration as the milipore filter of 50kD then; Collect ultrafiltrate, use the 1kD milipore filter to carry out ultrafiltration to trapped fluid again ultrafiltrate and be 200mL, the trapped fluid of gained is the antibacterial peptide biopesticide.
The application process of earthworm antibacterial peptide biopesticide: at rice seedling, get 300 times of the said goods 150mL thin ups, be mixed with soup, carry out stems and leaves of rice and spray.
Embodiment 3
The preparation of earthworm antibacterial peptide biopesticide: get earthworm 1000g alive and in water, soak 4h, wash foreign material after, use distilled water flushing again 3 times, put into the tank that positive and negative electrode is installed and links to each other with controller; Add 2000mL phosphate buffer (pH5.8), the liquid level of buffer solution is higher than electrode, and energized is under the 15V at voltage; To earthworm electro photoluminescence 1.5min, the earthworm of irriate is discharged juice and gets into phosphate buffer, and earthworm is taken out, and rubs with tissue refiner; Add 6000mL phosphate buffer (pH5.8), light and slow stirring and leaching 4h is heated to 80 ℃ again, insulation 15min; After the cooling, with 4000r/m, centrifugal 15min, it is subsequent use to obtain centrifugate; Buffer solution and above-mentioned centrifugate that other takes out in the tank merge, and with the membrane filtration of 1 μ m, collect filtrating, use molecular cut off to carry out ultrafiltration as the milipore filter of 50kD then; Collect ultrafiltrate, use the 1kD milipore filter to carry out ultrafiltration to trapped fluid again ultrafiltrate and be 250mL, the trapped fluid of gained is the antibacterial peptide biopesticide.
The application process of earthworm antibacterial peptide biopesticide: in the vegetative period of Chinese cabbage, get 400 times of the said goods 200mL thin ups, be mixed with soup and carry out the Chinese cabbage foliage-spray.
The above content is merely the basic explanation of the present invention under conceiving, and according to any equivalent transformation that technical scheme of the present invention is done, all should belong to protection scope of the present invention.
Claims (3)
1. earthworm antibacterial peptide biopesticide and application is characterized in that the preparation method of this agricultural chemicals comprises: earthworm is put into a tank that has positive and negative electrode and link to each other with controller, add the phosphate buffer (pH5.8～6.0) of 2 times of volumes; Energized is under voltage 15～20V, to earthworm electro photoluminescence 0.5～1.5min; Earthworm is taken out, earthworm is rubbed, add 6-8 times of volume phosphate buffer (pH5.8～6.0) with tissue refiner; Lixiviate 2-4h is heated to 80 ℃ again, insulation 15min; After the cooling, centrifugation, it is subsequent use to obtain centrifugate; Buffer solution and above-mentioned centrifugate that other takes out in the tank merge, and with the membrane filtration of 1 μ m, collect filtrating; Use molecular cut off to carry out ultrafiltration as the milipore filter of 50kD then, collect ultrafiltrate, the trapped fluid that uses the 1kD milipore filter to carry out ultrafiltration collection trapped fluid gained again ultrafiltrate is the antibacterial peptide biopesticide.
2. a kind of earthworm antibacterial peptide biopesticide according to claim 1 and application are to be used to spray plant stem-leaf or seed soaking in case the powdery mildew of harness the river rice rice blast, banded sclerotial blight, rice leaf roller, rice fulgorid, gourd, fruit and vegetable, gray mold, anthracnose and budworm are sick.
3. a kind of earthworm antibacterial peptide biopesticide according to claim 1 and application; It is characterized in that, with 200～400 times of uses of earthworm antibacterial peptide biopesticide dilute with water, when being used for paddy rice; Carrying out cauline leaf in seedling stage, vegetative period, flowering stage, pustulation period respectively sprays; When being used for gourd, fruit and vegetable, carrying out cauline leaf in seedling stage, vegetative period, flowering stage, fruit expanding period respectively and spray, the solution of 200～400 times of dilutions is adopted in paddy rice, gourd, fruit and vegetable seed soaking.
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| CN2010102785658A CN102396539A (en) | 2010-09-10 | 2010-09-10 | Earthworm antimicrobial peptide biological pesticide and application |
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| CN2010102785658A CN102396539A (en) | 2010-09-10 | 2010-09-10 | Earthworm antimicrobial peptide biological pesticide and application |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105272479A (en) * | 2015-11-19 | 2016-01-27 | 扬州大学 | Earthworm amino acid liquid fertilizer special for watermelons and producing method |
| CN109168802A (en) * | 2018-08-08 | 2019-01-11 | 上海市农业科学院 | A kind of method of biology prevention and control rice field disease |
| CN109260231A (en) * | 2017-07-18 | 2019-01-25 | 首都儿科研究所 | The preparation method of the anti-inflammatory antimicrobial extract of cough-relieving apophlegmatic in a kind of earthworm |
| CN109287887A (en) * | 2018-09-26 | 2019-02-01 | 何秋宏 | A kind of feed of grass carp and preparation method thereof |
| CN109749961A (en) * | 2019-02-20 | 2019-05-14 | 金正大生态工程集团股份有限公司 | A kind of microbial bacterial agent and the preparation method and application thereof for repairing garlic continuous cropping soil |
| CN112679573A (en) * | 2021-01-21 | 2021-04-20 | 广东莞垄农业生态科技有限公司 | Preparation method and application of earthworm antibacterial peptide |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1360833A (en) * | 2000-12-29 | 2002-07-31 | 中国农业大学 | Prepn for earthworm antibacterial peptide |
-
2010
- 2010-09-10 CN CN2010102785658A patent/CN102396539A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1360833A (en) * | 2000-12-29 | 2002-07-31 | 中国农业大学 | Prepn for earthworm antibacterial peptide |
Non-Patent Citations (2)
| Title |
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| 徐国珍: "蚯蚓抗菌肽的提取纯化及其对植物病原菌抑菌作用的初步研究", 《中国学位论文全文数据库》, 8 April 2004 (2004-04-08), pages 9 - 24 * |
| 王庄等: "蚯蚓中3-30KD抗菌肽的分离及抗菌效果研究", 《湖南农业大学学报（自然科学版）》, vol. 36, no. 1, 30 June 2010 (2010-06-30), pages 50 - 53 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105272479A (en) * | 2015-11-19 | 2016-01-27 | 扬州大学 | Earthworm amino acid liquid fertilizer special for watermelons and producing method |
| CN109260231A (en) * | 2017-07-18 | 2019-01-25 | 首都儿科研究所 | The preparation method of the anti-inflammatory antimicrobial extract of cough-relieving apophlegmatic in a kind of earthworm |
| CN109260231B (en) * | 2017-07-18 | 2021-09-03 | 首都儿科研究所 | Preparation method of earthworm extract with cough stopping, phlegm eliminating, anti-inflammatory and antimicrobial functions |
| CN109168802A (en) * | 2018-08-08 | 2019-01-11 | 上海市农业科学院 | A kind of method of biology prevention and control rice field disease |
| CN109168802B (en) * | 2018-08-08 | 2022-01-14 | 上海市农业科学院 | Method for biologically preventing and controlling rice field diseases |
| CN109287887A (en) * | 2018-09-26 | 2019-02-01 | 何秋宏 | A kind of feed of grass carp and preparation method thereof |
| CN109749961A (en) * | 2019-02-20 | 2019-05-14 | 金正大生态工程集团股份有限公司 | A kind of microbial bacterial agent and the preparation method and application thereof for repairing garlic continuous cropping soil |
| CN109749961B (en) * | 2019-02-20 | 2022-10-04 | 金正大生态工程集团股份有限公司 | Microbial agent for repairing garlic continuous cropping soil and preparation method and application thereof |
| CN112679573A (en) * | 2021-01-21 | 2021-04-20 | 广东莞垄农业生态科技有限公司 | Preparation method and application of earthworm antibacterial peptide |
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Application publication date: 20120404 |