CN100346699C - Adsorbent type of bacillus thuringlensis and preparation method - Google Patents
Adsorbent type of bacillus thuringlensis and preparation method Download PDFInfo
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- CN100346699C CN100346699C CNB2005100152823A CN200510015282A CN100346699C CN 100346699 C CN100346699 C CN 100346699C CN B2005100152823 A CNB2005100152823 A CN B2005100152823A CN 200510015282 A CN200510015282 A CN 200510015282A CN 100346699 C CN100346699 C CN 100346699C
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Abstract
The present invention discloses an adsorbent formulation of bacillus thuringiensis and a preparation method thereof. The adsorbent formulation uses soil-mineral micron-particles as carriers to absorb and fix pesticidal proteins after the solvent treatment, and the mass ratio of the pesticidal proteins to the soil-mineral particles is from 1: 50 to 5: 1; with the preparation method of the present invention, the pesticidal protein solution, the soil-mineral micron-particles and the soil particle suspension liquid are prepared through purifying and extracting the pesticidal proteins so that products are obtained through the adsorption of the pesticidal proteins. The method of the present invention for preparing the adsorbent formulation of the bacillus thuringiensis is simple and convenient, materials are easy to obtain, and the obtained adsorbent formulation has the advantages of high pesticidal activity, good particle dispersal, firm adsorption, resistance for ultraviolet degradation, and no generation for environmental safety problem; because the commercial formulation is used on the surface of crops, the degradation speed is higher than that of the adsorbed proteins in the soil, and the residual period can be controlled in the reasonable range through controlling the adsorption quantity of the pesticidal proteins in the adsorbent formulation and the formulation consumption.
Description
[technical field]
The present invention relates to the biological control technical field of pest, particularly a kind of microbial pesticide adsorbent type of bacillus thuringlensis and preparation method.
[background technology]
Bacillus thuringiensis,Bt (Bacillus thuringiensis, be called for short Bt) is a kind ofly to produce parasporal crystal, can parasitize the bacillus that causes the insect morbidity in the insect bodies.Since Japanese stone in 1901 crossed the report bacillus thuringiensis,Bt, the scientific research personnel had carried out broad research to structure, insect-resistant transgenic plants and the industrialization etc. of the biochemistry of its classification, growth metabolism, genetics, ecology, parasporal crystal toxin, mechanism of action, genetic engineering bacterium both at home and abroad.More than 120 kind registered by existing more than 60 country up to now, its output accounts for 90%～98% of biological insecticides total amount, become output maximum on the our times, most widely used, also be the most rising a kind of biological insecticides, be widely used in control agricultural, forestry, storage insect and medical insect.
Yet thuricade-1 exists the lasting period short, and control efficiency is subject to shortcomings such as extraneous weather such as sunlight, temperature, humidity, rainwater and ecological condition restriction.Common bacillus thuringiensis,Bt commercial formulation contains parasporal crystal, brood cell and filler, and wherein parasporal crystal is main Pesticidal toxins.Thuringiensis praeparatum preparation is after using on the crops, and the parasporal crystal toxin can be passivated rapidly and degrade, and the brood cell can not existence well in natural soils.According to foreign study, the ThuricideA preparation indoor use 8 days or 14 days after, it is initial 1/14 and 1/71 that virulence is reduced to respectively, the field test longevity of residure was generally 5-7 days.It is short that the longevity of residure is used in the bacillus thuringiensis,Bt field, become the bottleneck of this insecticide development of restriction, and how improving its insecticidal effect is the difficult problem that bacillus thuringiensis,Bt R﹠D institution and manufacturing enterprise are faced jointly.Prolonging the method in the longevity of residure at present mainly contains: add ultra-violet absorber, preparation microcapsules, composite with chemical pesticide.These methods have certain actual effect, but the desirable means that do not become a kind of economy, environmental protection as yet, efficiently take into account.
It is relevant with following three kinds of factors whether thuringiensis bacillus toxin can accumulate in environment: the concentration that use (1); (2) speed of insect consumption; (3) ultraviolet light and soil, crop surface micropopulation are to the speed of its passivation and degraded.Kind of crop successively enters commercialization production surplus the first the commentaries on classics after thuringiensis bacillus crystallin gene pest-resistant plant field trial succeeds from 1987, insect-resistant transgenic cotton, potato, corn, paddy rice, tobacco, tomato etc. 50.The trans Bt gene plant is continual in vegetative period to discharge Pesticidal toxins to environment, and often only gathers in the crops the part that it has economic worth after the crop maturation, and the straw that major part contains toxin is discarded into the field.Therefore, the toxin that genetically modified crops produce is often higher than the concentration of bacillus thuringiensis,Bt commercial formulation in soil, and it is more lasting to act on.Discover that the toxin that genetically modified crops discharge will produce resistance to microbial decomposition in case adsorbed by the soil particle in the environment, be adsorbed the sustainable existence of insecticidal activity 180 days or 234 days of albumen.Infrared spectrum and X-ray diffraction the analysis showed that the structure of absorption back toxin protein only has minor variations, and has good resistance to microbial degradation energy and insecticidal activity.Therefore, the experimental evidence that Pesticidal toxins was prolonged by the soil mineral absorption back longevity of residure is for new way has been opened up in the research of bacillus thuringiensis,Bt durative action preparation.
[summary of the invention]
The objective of the invention is to overcome the existing existing defective of microbial pesticide bacillus thuringiensis,Bt formulation, and a kind of adsorbent type of bacillus thuringlensis and preparation method are provided.This method is extracted Bacillus thuringiensis insecticidal proteins, be dissolved in the alkaline solution behind the purifying, and it is fixing with insecticidal proteins absorption to add the soil mineral particulate again, thereby obtains stable performance, adsorbent type of bacillus thuringlensis that insecticidal activity is high.
For realizing purpose of the present invention, the invention discloses a kind of adsorbent type of bacillus thuringlensis.It is characterized in that said adsorbent type is a carrier with micron-sized soil mineral particle, absorption is fixed with the insecticidal proteins after the dissolution process on the carrier, and the mass ratio of insecticidal proteins and soil mineral particle is: 1: 50-5: 1.
The invention also discloses the preparation method of this adsorbent type of bacillus thuringlensis, it is characterized in that operating according to the following step:
(1) the former powder of thuringiensis is washed respectively with NaCl solution and aseptic deionized water after, extracted 4-5 hour with the NaOH solution of pH11-12, the centrifugal post precipitation that goes obtains the insecticidal proteins aqueous slkali, the acetate precipitation.Insecticidal proteins is dissolved in the aqueous slkali that pH is 9-11, is configured to the aqueous slkali of concentration 0.05%-0.25% (W/V).
(2) preparation micron order soil mineral particle: will add the suspension that deionized water is made into solid content 1.0%-10.0% (W/V) through the dry soil that grinds and cross 100 mesh sieves, fully stir the back sedimentation, take out the upper strata suspension after the sedimentation then, repeat aforesaid operations, upper strata suspension clarification after sedimentation.The upper strata suspension that collect to take out with the flocculation of 0.5mol/L calcium chloride, regathers precipitation, and the oven dry back grinds, and obtains the particle of discrete particles in water＜2 micron;
(3) preparation soil particle suspension: get the suspension that micron order soil mineral particle water is configured to 1.0%-10.0% (W/V);
(4) insecticidal proteins adsorption treatment on soil particle: in the mass ratio of insecticidal proteins and soil mineral particle: 1: 50-5: 1 ratio, temperature-10 ℃-50 ℃, absorption is 0.5-10 hour under the shaking table jolting condition;
(5) absorption is centrifugal after finishing deviates from supernatant, is soil mineral granule adsorbent type, freezing preservation.
The method that the present invention prepares adsorbent type of bacillus thuringlensis is easy, and material is easy to get.Resulting adsorbent type has following characteristics:
(1) insecticidal activity height.Tire the bacillus thuringiensis,Bt wetting powder of 20000IU/mg to the median lethal concentration (LC of cotton bollworm third-instar larvae
50) be 38.77 μ g/ml, and the LC of soil micelle adsorbent type
501.02～3.36 μ g/ml (in the insecticidal proteins that is adsorbed, as follows), the latter's insecticidal activity improves 11～38 times than the former.With the pure insecticidal proteins (LC that is not adsorbed
50Be 10 μ g/ml) to compare, the insecticidal activity of soil micelle adsorbent type improves 3～10 times;
(2) particle is scattered.Particle size analysis does not have significant change before and after the soil micelle absorption insecticidal proteins, and in water good dispersion property;
(3) absorption firmly.The insecticidal proteins that is adsorbed by the soil micelle is not easy to be separated by water and carbonate buffer solution and sops up.Deionized water continuous desorption three times, total desorption efficiency is 24.66%～40.54%; Carbonate buffer solution continuous desorption three times, total desorption efficiency is 15.37%～38.81%;
(4) ultraviolet light degraded.Through UV-irradiation 1 hour, the former powder insecticidal activity of bacillus thuringiensis,Bt descended 21.55%, and the pure toxin that is not adsorbed descends 23.66%, and the bentone adsorbent type only descends 8.11%, and the Red soil colloid adsorbent type descends 14.07%.
In addition, this preparation is not only prolonged by the insecticidal proteins of adsorption by soil action time, insecticidal activity improves, and use can not produce the Environmental security problem yet, because commercial formulation is to use in crop surface, its degradation speed by the adsorbing capacity of insecticidal proteins and the using dosage of preparation in the control absorption preparation, just can be controlled at reasonable range with its longevity of residure than fast at the albumen of the inner absorption of soil.
[embodiment]
A kind of adsorbent type of bacillus thuringlensis of the present invention is a carrier with micron-sized soil mineral particle, and absorption is fixed with the insecticidal proteins of dissolved state on the carrier, and the mass ratio of insecticidal proteins and soil mineral particle is: 1: 50-5: 1.Above-mentioned soil mineral can be southern red soil, red earth, laterite, yellow earth, yellowish soil, the brown earth in the north, cold brown earth, brown soil, dark brown earth, cinnamon soil, chernozem, western black mature soil and alpine meadow soil, and one or both or two or more mixtures in the bentonite, kaolin.
A kind of bacillus thuringiensis,Bt adsorbent of the present invention can prepare by the following method:
(1) preparation of thuringiensis insecticidal proteins:
Get the former powder of thuringiensis with 0.5mol/L NaCl washing three times, wash three times with aseptic deionized water, 10000r/min is centrifugal, and 10min removes supernatant.Precipitation is made into the aqueous suspensions of 1%-5% (W/V), transfers pH11-12 with 0.5-1mol/L NaOH, the centrifugal precipitation of going after 4-5 hour, clear liquid is transferred pH4.4 with 25% acetate, and the centrifugal 10min of 15000r/min gets the insecticidal activity albumen precipitation.
(2) preparation of Bacillus thuringiensis insecticidal proteins solution:
The thuringiensis insecticidal proteins is dissolved in aqueous slkali, and aqueous slkali comprises sodium hydroxide solution, potassium hydroxide solution, sodium carbonate liquor, solution of potassium carbonate, Huber buffer solution (0.1mol/LNa
2CO
3-HCl contains the 25mmol/L dithiothreitol (DTT), pH9.5), but preferably adopts carbonate buffer solution (being made up of carbonate and bicarbonate).Alkali lye pH value is preferably between the 9.5-10.5 between 9-11.Insoluble composition adopts centrifugal process to remove, and insecticidal proteins concentration is between 0.05%-0.25% (W/V) in the alkali lye, and concentration value adopts determined by ultraviolet spectrophotometry.
(3) preparation of little next level soil mineral particle:
Earth sample such as southern red soil, red earth, laterite, yellow earth, yellowish soil fetch earth, the brown earth in the north, cold brown earth, brown soil, dark brown earth, cinnamon soil, chernozem, western black mature soil and alpine meadow soil, and one or both or two or more mixtures in the bentonite, kaolin, add the suspension that water is made into solid content 1%-10% (W/V), but be preferably 1%～5%.Suspension is changed in the beaker, liquid level keeps 15cm, fully stir back free settling, calculate the diameter needed time of the 10cm of soil particle sedimentation below 2 microns according to Stokes sedimentation law, and on schedule from the above sucking-off of beaker bottom 5cm upper strata suspension.Aforesaid operations carries out repeatedly, till suspension clarification in upper strata after the sedimentation.Collect the upper strata suspension that takes out, with the flocculation of 0.5mol/L calcium chloride.Collecting precipitation, oven dry back grinds, and crosses 100 mesh sieves, promptly gets the soil specimen of discrete particles in water＜2 micron.
(4) preparation of soil particle suspension:
Get the suspension that above-mentioned micron order soil mineral particle water is configured to 1.0%-10.0% (W/V), be preferably 1.0%-5.0%, ultrasonic disperses the back standby.The used water of preparation soil supension is distilled water or deionized water, but preferably adopts aseptic deionized water.
(5) absorption of Bacillus thuringiensis insecticidal proteins:
With concentration is that the insecticidal proteins solution of 0.1%-0.25% (W/V) places in the 100ml plastic tube, and the adding volume of soil supension calculates according to the mass ratio of insecticidal proteins of stipulating below and soil particle.The mass ratio of insecticidal proteins and soil micelle is controlled at 1: 50-5: between 1, but preferably be controlled at 1: 5-2: between 1.Under temperature-10 ℃-50 ℃, change constant temperature (25 ℃) shaking table over to after even shaking on the oscillator, jolting speed 100-300r/min took out after 0.5-10 hour, the centrifugal 20min of 18000r/min on supercentrifuge, absorption is finished.Change supernatant over to another sterile test tube, adopt ultraviolet absorption method to measure absorbance in the 280nm place,, can calculate the insecticidal proteins amount of soil particle absorption according to the variation of insecticidal proteins concentration before and after the absorption.
(6) soil micelle-insecticidal proteins compound is placed refrigerator ,-18 ℃ of freezing preservations.
Embodiment 1: the bentone adsorbent type
Get former powder 20 grams of thuringiensis, with 0.5mol/L NaCl washing three times, with aseptic deionized water washing three times, 10000r/min is centrifugal, and 10min removes supernatant again.Precipitation is made into 5% aqueous suspensions, transfers pH11-12 with 1mol/L NaOH, the centrifugal precipitation of going after 4-5 hour, clear liquid is transferred pH4.4 with 25% acetate, and the centrifugal 10min of 15000r/min gets insecticidal activity albumen precipitation 1.3 grams.Insecticidal proteins 1.3 grams are dissolved in 650ml sodium carbonate buffer (pH9.5), through determined by ultraviolet spectrophotometry, insecticidal proteins concentration 1.93mg/ml.
Bentonite is selected from Xinyang, Henan.50 gram soil specimens are suspended in the 2000ml water, make bentonite clay particle 3.5 grams of particle diameter＜2 micron, get 1 gram with aseptic deionized water configuration 1% (W/V) suspension through the sedimentation centrifugal process.The adsorption experiment of insecticidal proteins is undertaken by 1: 1 (mass ratio) according to insecticidal proteins and bentonite.The insecticidal proteins carbonate solution of 20ml concentration 1.93mg/ml is added in the 100ml plastic test tube (containing pure protein 38.6mg), add concentration 1% bentonite suspension 3.86ml (containing bentonite 38.6mg), add carbonate buffer solution (pH9.5) 15ml and dilute.Take out behind constant temperature shaking table (25 ± 1 ℃) the 300r/min jolting 5h, the centrifugal 10min of 18000r/min, the supernatant that inclines gets bentonite-insecticidal proteins compound.The LC of bentonite-insecticidal proteins compound (being the bentone adsorbent type)
50Be 1.02 μ g/ml.Behind the ultraviolet lighting 1 hour, LC
50Be 1.11 μ g/ml, descend 8.11%.
Embodiment 2: the bentone adsorbent type
Sterile water suspension with bentonite micelle configuration 5% (W/V) of particle diameter＜2 micron.Insecticidal proteins is mixed by 1: 5 (mass ratio) with bentonite, take out behind constant temperature shaking table (25 ± 1 ℃) the 300r/min jolting 3h, the centrifugal 10min of 18000r/min, supernatant inclines.The LC of bentone adsorbent type
50Be 2.12 μ g/ml.
Embodiment 3: the bentone adsorbent type
Sterile water suspension with bentonite micelle configuration 3% (W/V) of particle diameter＜2 micron.Insecticidal proteins is mixed by 2: 1 (mass ratio) with bentonite, take out behind constant temperature shaking table (25 ± 1 ℃) the 300r/min jolting 1h, the centrifugal 10min of 18000r/min, supernatant inclines.The LC of bentone adsorbent type
50Be 1.93 μ g/ml.
Embodiment 4: the Red soil colloid adsorbent type
Red soil is selected from Chenzhou, Hunan.Soil specimen is suspended in water, make the particle of particle diameter＜2 micron, sterile water configuration 1% (W/V) suspension through the sedimentation centrifugal process.Insecticidal proteins is mixed in 1: 10 (mass ratio) ratio with bentonite, take out behind constant temperature shaking table (25 ± 1 ℃) the 300r/min jolting 0.5h, the centrifugal 10min of 18000r/min, supernatant inclines.The LC of Red soil colloid adsorbent type
50Be 3.36 μ g/ml.Behind the ultraviolet lighting 1 hour, LC
50Be 3.91 μ g/ml, descend 14.07%.
Embodiment 5: the kaolin adsorbent type
Kaolin is purchased in Shanghai the May 4th chemical reagent factory.Soil specimen is suspended in water, make the particle of particle diameter＜2 micron, sterile water configuration 1% (W/V) suspension through the sedimentation centrifugal process.Insecticidal proteins is mixed in 1: 1.25 (mass ratio) ratio with bentonite, take out behind constant temperature shaking table (25 ± 1 ℃) the 300r/min jolting 3h, the centrifugal 10min of 18000r/min, supernatant inclines.The LC of kaolin adsorbent type
50Be 2.33 μ g/ml.
Embodiment 6: the yellowish soil adsorbent type
Yellowish soil is selected from Yingcheng City, Hubei.Soil specimen is suspended in water, make the micelle of particle diameter＜2 micron, sterile water configuration 10% (W/V) suspension through the sedimentation centrifugal process.Insecticidal proteins is mixed in 1: 15 (mass ratio) ratio with yellowish soil, take out behind constant temperature shaking table (25 ± 1 ℃) the 300r/min jolting 3h, the centrifugal 10min of 18000r/min, supernatant inclines.The LC of yellowish soil adsorbent type
50Be 3.02 μ g/ml.
Claims (11)
1. adsorbent type of bacillus thuringlensis, it is characterized in that said adsorbent type is a carrier with micron-sized soil mineral particle, absorption is fixed with the insecticidal proteins after the dissolution process on the carrier, and the mass ratio of insecticidal proteins and soil mineral particle is: 1: 50-5: 1; The diameter of described micron order soil mineral particle is less than 2 microns.
2. according to the described adsorbent type of bacillus thuringlensis of claim 1, it is characterized in that said soil mineral is southern red soil, red earth, laterite, yellow earth, yellowish soil, the brown earth in the north, cold brown earth, brown soil, dark brown earth, cinnamon soil, chernozem, western black mature soil and alpine meadow soil, and one or both or two or more mixtures in the bentonite, kaolin.
3. the preparation method of the described adsorbent type of bacillus thuringlensis of claim 1 is characterized in that operating according to the following step:
(1) the former powder of thuringiensis is washed respectively with NaCl solution and aseptic deionized water after, extracted 4-5 hour with the NaOH solution of pH11-12, the centrifugal post precipitation that goes obtains the insecticidal proteins aqueous slkali, the acetate precipitation; Insecticidal proteins is dissolved in the aqueous slkali that pH is 9-11, is configured to the aqueous slkali of concentration 0.05%-0.25% (W/V);
(2) preparation micron order soil mineral particle: will add the suspension that deionized water is made into solid content 1.0%-10.0% (W/V) through the dry soil that grinds and cross 100 mesh sieves, fully stir the back sedimentation, take out the upper strata suspension after the sedimentation then, repeat aforesaid operations, upper strata suspension clarification after sedimentation; The upper strata suspension that collect to take out with the flocculation of 0.5mol/L calcium chloride, regathers precipitation, and the oven dry back grinds, and obtains the particle of in water discrete particles diameter＜2 micron;
(3) preparation soil particle suspension: get the suspension that micron order soil mineral particle water is configured to 1.0%-10.0% (W/V);
(4) insecticidal proteins adsorption treatment on soil particle: in the mass ratio of insecticidal proteins and soil mineral particle: 1: 50-5: 1 ratio, temperature-10 ℃-50 ℃, absorption is 0.5-10 hour under the shaking table jolting condition;
(5) absorption is centrifugal after finishing deviates from supernatant, is soil mineral granule adsorbent type, freezing preservation.
4. in accordance with the method for claim 3, the aqueous slkali that it is characterized in that said step (1) dissolving insecticidal proteins is sodium hydroxide solution, potassium hydroxide solution, sodium carbonate liquor, solution of potassium carbonate, Huber buffer solution, carbonate buffer solution, and alkali lye pH is at 9.5-10.5.
5. according to claim 3 or 4 described methods, it is characterized in that the suspension that said step (3) is mixed with soil particle, solid content is 1.0%-5.0% (W/V); Institute's water is distilled water or deionized water, also can be aseptic deionized water.
6. according to claim 3 or 4 described methods, the mass ratio that it is characterized in that said step (4) insecticidal proteins and soil mineral particle is 1: 5-2: 1.
7. in accordance with the method for claim 5, the mass ratio that it is characterized in that said step (4) insecticidal proteins and soil mineral particle is 1: 5-2: 1.
8. according to claim 3 or 4 described methods, it is characterized in that the adsorption temp of said step (4) insecticidal proteins is-4 ℃-30 ℃, adsorption time is 0.5-3 hour.
9. in accordance with the method for claim 5, it is characterized in that the adsorption temp of said step (4) insecticidal proteins is-4 ℃-30 ℃, adsorption time is 0.5-3 hour.
10. in accordance with the method for claim 6, it is characterized in that the adsorption temp of said step (4) insecticidal proteins is-4 ℃-30 ℃, adsorption time is 0.5-3 hour.
11. in accordance with the method for claim 7, it is characterized in that the adsorption temp of said step (4) insecticidal proteins is-4 ℃-30 ℃, adsorption time is 0.5-3 hour.
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| CN101946824A (en) * | 2010-08-10 | 2011-01-19 | 湖南省粮油科学研究设计院 | Single-crystal grain storage protective agent used for grain pest prevention and preparation method |
| CN102038005B (en) * | 2010-12-15 | 2013-03-20 | 天津农学院 | Method for enhancing adsorption of Bacillus thuringiensis insecticidal proteins on mineral carrier |
| CN102038006B (en) * | 2010-12-15 | 2013-03-20 | 天津理工大学 | Preparation method of bacillus thuringiensis insecticidal protein and spore endospore adsorption composite dosage form |
| CN105076211B (en) * | 2014-05-07 | 2018-06-22 | 华中农业大学 | A kind of nanometer of Bt compounded pesticides and preparation method and application |
| CN113662016B (en) * | 2021-09-02 | 2022-08-09 | 中国农业科学院植物保护研究所 | Granules, preparation method and application thereof |
| CN115197022A (en) * | 2022-07-19 | 2022-10-18 | 深圳康民农业生物科技发展有限公司 | Organic fertilizer micron-coated slow release method |
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| CN1059830A (en) * | 1991-07-29 | 1992-04-01 | 商丘市医药化工厂 | The one plant species production method of bacterium for killing insect |
| CN1347647A (en) * | 2000-10-08 | 2002-05-08 | 孙家隆 | Pesticide composition |
| US6387386B1 (en) * | 1995-03-17 | 2002-05-14 | Lee County Mosquito Control District | Controlled delivery compositions and processes for treating organisms in a column of water or on land |
| CN1463604A (en) * | 2002-06-03 | 2003-12-31 | 杨爱民 | Pest control agent comprising multiple micro-organism |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1043603A (en) * | 1988-12-24 | 1990-07-11 | 湖南省微生物研究所 | Granular mixture of thuringiensis bacillus |
| CN1059830A (en) * | 1991-07-29 | 1992-04-01 | 商丘市医药化工厂 | The one plant species production method of bacterium for killing insect |
| US6387386B1 (en) * | 1995-03-17 | 2002-05-14 | Lee County Mosquito Control District | Controlled delivery compositions and processes for treating organisms in a column of water or on land |
| CN1347647A (en) * | 2000-10-08 | 2002-05-08 | 孙家隆 | Pesticide composition |
| CN1463604A (en) * | 2002-06-03 | 2003-12-31 | 杨爱民 | Pest control agent comprising multiple micro-organism |
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