CN112970782A - Pesticide preparation obtained by fermenting streptomyces lavipediti and preparation method thereof - Google Patents
Pesticide preparation obtained by fermenting streptomyces lavipediti and preparation method thereof Download PDFInfo
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- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
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- 235000020232 peanut Nutrition 0.000 description 1
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- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
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Abstract
The invention discloses a pesticide preparation obtained by fermenting Lauyidede streptomycete and a preparation method thereof, wherein the Lauyidede streptomycete strain is obtained by a physical mutagenesis method, the strain obtained by separating a plurality of soils is screened to obtain an actinomycete strain, the physical mutagenesis is carried out, secondary metabolites have better control effect on sucking mouthpart pests, the strain is subjected to gene identification, and the strain is confirmed to be the Lauyidede streptomycete. The strain is fermented and post-treated to obtain raw powder, and can be made into wettable powder and water dispersible granule. The preparation can be used for preventing and treating sucking mouthpart pests such as aphid and thrips.
Description
Technical Field
The invention relates to the technical field of pesticide preparations, and particularly relates to a pesticide preparation obtained by fermenting streptomyces laveriensis and a preparation method thereof.
Background
Sucking mouthparts pests, such as aphids, spider mites, plant hoppers, and the like. Their relative classification status is very different, but the hazard patterns are very similar. They use a slender needle-shaped mouthpart to prick and suck nutrient juice in the surface of a plant (including leaves, flowers, fruits and peels), so that cells are damaged, the growth is unbalanced, the plant stops growing when the plant is serious, and even the whole plant wilts. Most piercing-sucking mouthpart pests are small in size, but are high in propagation speed, extremely large in quantity, long in population damage period and many in generations all year round. Meanwhile, the virus is a carrier of the virus and an intermediate medium, can cause the crop to be infected with the virus disease, and seriously influences the agricultural production.
In practice, chemical agents are mostly used for preventing and controlling pests such as aphids, thrips, mites and the like, such as imidacloprid, acetamiprid, nitenpyram, thiamethoxam and the like, some pests are easy to generate resistance, and some pests have long pesticide residue time. An environment-friendly biological medicament for preventing and treating the pests with the piercing-sucking mouthparts is urgently needed in agricultural production.
Disclosure of Invention
The invention aims to provide a pesticide preparation obtained by fermenting streptomyces lavipediti and a preparation method thereof, and a strain with biological activity is obtained by performing biological test on strains separated from a plurality of soils. Selecting nitrogen atoms and boron atoms with different energy levels to impact bacterial suspension of a preferred strain, screening to obtain a high-yield actinomycete strain, finding that a secondary metabolite of the actinomycete strain has a good control effect on the sucking mouthpart pests, carrying out gene identification on the strain, and confirming that the strain is the Laurede streptomycete. Biopesticides of microbial origin generally contain a variety of components, possibly involving a variety of mechanisms of action, and are not susceptible to resistance, in order to solve the problems set forth in the background art above.
In order to achieve the purpose, the invention provides the following technical scheme:
collecting a soil sample, separating a plurality of actinomycete strains, fermenting by using a Gauss culture medium, performing biological test on the obtained fermentation liquor, and screening to obtain the control effect of the actinomycete strain on the pests with the piercing-sucking mouthparts. The strain is subjected to physical mutagenesis, nitrogen atoms and boron atoms with different energy levels are selected to impact the strain suspension of the optimized strain, and a high-yield actinomycete strain is obtained by screening, so that the control effect on piercing-sucking mouthpart pests is obviously improved. The strain is identified to be the strain Streptomyces lavendulae.
A pesticide preparation is prepared by fermenting Streptomyces laveliensis, wherein the strain of Streptomyces laveliensis is obtained by physical mutagenesis.
The other technical scheme to be solved by the invention is as follows: a preparation method of a pesticide preparation obtained by fermenting streptomyces laveriensis comprises the following steps:
s101: inoculating streptomyces lavipediti into a seed tank, and culturing for about 24 hours to obtain seed liquid;
s102: inoculating the obtained seed liquid into a fermentation tank, and carrying out aerobic fermentation to obtain fermentation liquor containing bioactive substances;
s103: and carrying out post-treatment on the obtained fermentation liquor to obtain raw powder.
Furthermore, the raw powder can be added with a wetting agent, a dispersing agent and a filling material to prepare wettable powder:
30-60% of raw powder, YP 5%, 5% of white carbon black, 10% of citric acid and 20-50% of kaolin.
Furthermore, the raw powder can be added with a wetting agent, a dispersing agent, an adhesive and a filler to prepare water dispersible granules:
30-60% of raw powder, 5% of castor oil polyoxyethylene ether, 3% of sodium dodecyl sulfate, 20% of corn starch and 12-42% of ammonium sulfate.
Further, the pesticide preparation can be used for preventing and controlling sucking mouthparts pests such as aphids, thrips, whiteflies and the like.
Compared with the prior art, the invention has the beneficial effects that: the pesticide preparation obtained by fermenting the streptomyces laveri and the preparation method thereof provided by the invention obtain a high-yield strain through physical mutagenesis, and secondary metabolite has a good control effect on pests with piercing-sucking mouthparts. Microbial biopesticides generally contain multiple components, may contain multiple mechanisms of action, and are not susceptible to resistance.
Drawings
FIG. 1 is a flow chart of the inoculation process in the seed tank of the present invention;
FIG. 2 is a flow chart of a sampling process of the fermenter according to the present invention;
FIG. 3 is a diagram showing a spore map of a medium in a fermenter according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A pesticide preparation obtained by fermenting streptomyces laveri comprises a culture medium of a Klebsiella inclined plane and a culture medium of a shake flask, wherein:
the culture medium for the inclined plane of the Kirschner flask comprises the following formula in parts by weight:
1.04% of corn extract, 0.3% of ammonium sulfate, 1% of soluble starch, 0.3% of calcium carbonate, 0.3% of sodium chloride and 1.8% of agar;
the fermentation medium comprises the following formula in parts by weight:
4.0% of peanut cake powder, 2.0% of glucose, 1.5% of corn starch, 0.3% of yeast powder, 0.36% of calcium carbonate, 0.2% of sodium chloride and 0.02% of dipotassium phosphate.
The PH value of the culture medium of the inclined plane of the Kirschner flask is 6.8-7.2, and the PH value of the fermentation culture medium is 6.7-7.0.
The shake flask filling amount of the shake flask culture medium is 30mL, and the culture temperature is as follows: 28 ℃, cycle: and (5) 24 h.
And (5) shaking the bottle for microscopic examination: hyphae grow vigorously for 24h, are interwoven into a net, and are free of mixed bacteria.
The passage method comprises the following steps: dipping the fermentation liquor in the shake bottle by a cotton swab and coating the fermentation liquor on the inclined surface of the kirschner bottle.
In order to better show the preparation process of the pesticide preparation obtained by fermenting streptomyces laveriensis, the embodiment provides a preparation method of the pesticide preparation obtained by fermenting streptomyces laveriensis, which comprises a pilot plant process, wherein the pilot plant process comprises an inoculation process in a seed tank and a sampling process of a fermentation tank, and the preparation method comprises the following steps:
as shown in FIG. 1, the inoculation process in the seeding tank includes the following steps:
s101: the seeding tank is 1 ton tank, the culture medium has constant volume of 0.5 ton, the culture medium formula is as above shake flask culture medium formula, and sterilization is carried out at 121 deg.C for 30 min;
s102: pouring about 30mL of sterile water into a Kirschner flask in a sterile environment, scraping off thalli by using a sterile cotton swab, and fully shaking;
s103: 1, inoculation amount: 50-100, preparing bacterial suspension by using a Kirschner flask, and connecting the bacterial suspension with 50-100L of liquid culture medium; the seeding tank inoculation amount is recommended to be 5 Kirschner bottles.
The process parameters of the process are as follows in table 1:
seeding tank transfer fermenter standard: the hyphae grow vigorously and are interwoven into a net without mixed bacteria.
Referring to FIG. 2, the sampling process of the fermenter comprises the following steps:
s201: the fermentation tank is a 5-ton tank, the volume of the culture medium is 3 tons, the formula of the culture medium is as that of the above shaking culture medium, and the sterilization is carried out for 30min at 121 ℃;
s202: tank-placing standard, Ph: 6.0, hyphae were broken and a large number of spores were produced, as shown in FIG. 3;
s203: sampling time: sampling every 8h, detecting the items of PH, amino nitrogen, total sugar, reducing sugar and bacterial concentration, and simultaneously recording the PH, dissolved oxygen, stirring, tank temperature and tank pressure every hour.
The process parameters of the process are as follows in table 2:
powder spraying: according to the solid content of the fermentation liquor, 0.3 percent of calcium carbonate is added, and powder is directly sprayed without concentration by adopting a workshop spray tower.
The raw powder can be prepared into wettable powder according to the following formula:
40% of raw powder, 5% of YP, 5% of white carbon black, 10% of citric acid and 40% of kaolin.
In a laboratory, the result shows that the pesticide preparation has higher biological activity against harmful piercing-sucking insects. Such as: aphids, red spiders, red fruit mites, thrips tabaci, california beetles, cotton bollworms and braunie. Under the condition of a laboratory, the wild pea aphid, the cotton aphid, the peach aphid and the pea aphid are treated by 0.125-1% of pesticide preparation suspension, and the death rate is 50-100% after 24 hours. The most sensitive to pesticide formulations are cotton aphids, green peach aphids. The dosage of a laboratory sample of the insecticidal effect of a phytophagous insect can be determined.
TABLE 3 lethal concentration (%) and probability analysis calculation of pesticide preparation
The strain of the Laurednerella has secondary metabolite A and B components, wherein the A component is hygroscopic brown powder with molecular weight of 1207. The composition has biological activity on pests such as aphid and thrips. Compared with the compounds known from the prior literature, the component A is considered to be a novel insecticidal and acaricidal agent. The B component has a distinct maximum absorption wavelength at 221nm in the UV spectrum and has a broad peak at 240-280 nm. The B component has 667 ion peaks in the mass spectrum. The composition has biological activity on pests with piercing-sucking mouthparts. Based on the comparison of the results of infrared spectroscopy and the like with known data, the B component was classified as threonine type peptidoglycan and considered as a novel compound.
The wettable powder treated wheat aphid field control effect test:
the test method comprises the following steps: the wheat is sprayed by a spraying method, the test medicament is diluted by adding water according to the dosage of 750 g and 900 g per hectare, each treatment is repeated in 4 plots, the plants in the plots are uniformly sprayed, the plots are randomly arranged, and the test medicament is sprayed by a knapsack electric compression sprayer until the water drops on the leaf surfaces. Clear water treatment was used as a blank control, and investigation was conducted 3 days and 7 days after application. Sampling at five points in each cell, fixing 5-10 aphids in each point, and adjusting the number of aphids on each point.
The calculation method of the population reduction rate and the prevention and treatment effect comprises the following steps:
the control effect of the pesticide preparation on wheat aphids is shown in table 4:
TABLE 4 prevention and treatment effects of agricultural chemical preparations obtained by fermentation of Streptomyces laviperidis on wheat aphids
The pesticide preparation obtained by fermenting the streptomyces lavipediti has low toxicity and low residue on bees, birds, fish and silkworms in the environment, but has good control effect on pests such as aphids, thrips and the like.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical solutions and the inventive concepts of the present invention within the technical scope of the present invention.
Claims (5)
1. A pesticide preparation obtained by fermenting the LauredeStreptomycete is characterized in that the LauredeStreptomycete strain is obtained by a physical mutagenesis method.
2. The process for the preparation of a pesticidal formulation obtained by fermentation of streptomyces laveri according to claim 1, comprising the steps of:
s101: inoculating streptomyces lavipediti into a seed tank, and culturing for about 24 hours to obtain seed liquid;
s102: inoculating the obtained seed liquid into a fermentation tank, and carrying out aerobic fermentation to obtain fermentation liquor containing bioactive substances;
s103: and carrying out post-treatment on the obtained fermentation liquor to obtain raw powder.
3. The process for the preparation of a pesticidal formulation from streptomyces laveri fermentation as claimed in claim 2, wherein the raw powder can be made into wettable powder by adding wetting agent, dispersing agent, filler:
30-60% of raw powder, YP 5%, 5% of white carbon black, 10% of citric acid and 20-50% of kaolin.
4. The method for preparing a pesticide preparation obtained by fermenting streptomyces laveri as claimed in claim 2, wherein the raw powder can be added with a wetting agent, a dispersing agent, a binding agent and a filler to prepare water dispersible granules:
30-60% of raw powder, 5% of castor oil polyoxyethylene ether, 3% of sodium dodecyl sulfate, 20% of corn starch and 12-42% of ammonium sulfate.
5. The process for the preparation of a pesticidal formulation obtained by fermentation of streptomyces laveri according to claim 2, wherein the pesticidal formulation is used for controlling sucking mouthparts pests such as aphids, thrips, whitefly and the like.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU3893493A (en) * | 1992-04-01 | 1993-11-08 | Aventis Pharma S.A. | Micro-organisms, preparation method therefor and uses thereof |
| CN1152338A (en) * | 1994-07-08 | 1997-06-18 | 罗纳-布朗克罗莱尔股份有限公司 | Streptogramines and method for preparing same by mutasynthesis |
-
2019
- 2019-12-16 CN CN201911295947.9A patent/CN112970782A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU3893493A (en) * | 1992-04-01 | 1993-11-08 | Aventis Pharma S.A. | Micro-organisms, preparation method therefor and uses thereof |
| CN1152338A (en) * | 1994-07-08 | 1997-06-18 | 罗纳-布朗克罗莱尔股份有限公司 | Streptogramines and method for preparing same by mutasynthesis |
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