CN105010404A - Insecticidal activity substance and preparation method and application thereof - Google Patents
Insecticidal activity substance and preparation method and application thereof Download PDFInfo
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- CN105010404A CN105010404A CN201510405611.9A CN201510405611A CN105010404A CN 105010404 A CN105010404 A CN 105010404A CN 201510405611 A CN201510405611 A CN 201510405611A CN 105010404 A CN105010404 A CN 105010404A
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Abstract
The invention provides a method for preparing an insecticidal activity substance from secondary metabolism products of long shoot trichoderma strains. The method comprises the following steps of: step 1: performing liquid fermentation on the long shoot trichoderma strains capable of producing the insecticidal activity substance so as to obtain a culture solution containing insecticidal activity metabolism products; and step 2: performing a crude extraction operation on the obtained culture solution containing the insecticidal activity metabolism products so as to obtain a crude extract of the insecticidal activity substance. The invention further provides the application of the insecticidal activity substance prepared by the preparation method in the respects of preventing and treating bemisia tabaci and strawberry red spiders. The insecticidal activity substance and the preparation method and the application thereof disclosed by the invention have the advantages that preventing and treating trialeurodes uaporariorum and the strawberry red spiders through the strains are safe and reliable, phytotoxicity is not produced, human health is not endangered, and environment is not polluted.
Description
Technical field
The present invention relates to biological control plant insect field, particularly relate to a kind of insecticide active substance, particularly, relate to a kind of insecticide active substance prepared from the secondary metabolite of long shoot Trichoderma, and the preparation method of this insecticide active substance and the application in biological control plant insect thereof, the application especially in Bemisia tabaci and strawberry spider are prevented and treated.
Background technology
Bemisia tabaci [Bemisia tabaci (Gennadius)], is commonly called as little Bai moth, is a kind of global insect.Be primary in Perenniporia martius district, nursery stock by flowers and other economic crops is spread rapidly, in wide-scale distribution all over the world and population outbreak, also become the de novo a kind of insect pest of China in recent years, numerous crops such as vegetables and cotton such as harm tomato, cucumber, capsicum.Bemisia tabaci directly stings suction water, causes plant weak, and nymph and adult can also secrete honeydew, bring out the generation of sooty mould, and during density height, blade presents black, has a strong impact on photosynthesis.In addition, Bemisia tabaci can also propagate the virus disease of more than 70 kinds on 30 kinds of crops, causes serious loss to agricultural production.Bemisia tabaci have host extensively, body by wax, generation overlap, the feature such as reproduction speed is fast, Spreading and diffusion approach is many, control is difficulty very.
Strawberry spider, formal name used at school Tetranychus cinnabarinus (Tetranychuscinnabarinus Boisduval) is the important pests of strawberry, strawberry is in the growth intermediary and later stages, what often suffer two spotted spider mite breaks out harm, and killed plant downgrades early ageing, and blade is that bronzing dries up.Be injured in flowering stage, then fruit reduces, and malformed fruit increases, and has a strong impact on the output of strawberry fresh fruit.
People use chemical pesticide disease and pest control always for a long time, but the toxicity of chemical pesticide is comparatively large and residual quantity is also higher, eats fruit raw especially, use chemical pesticide for a long time as strawberry is this kind of, all can produce the health of environment and people and have a strong impact on, also can destroy the ecological balance.So need to screen further and develop, effectively prevent and treat the biopesticide of efficient, the low toxicity of Bemisia tabaci and two spotted spider mite, in order to reduce and the usage amount of substituting chemical pesticide.
Summary of the invention
Based on above-mentioned background technology, the invention provides a kind of insecticide active substance prepared from the secondary metabolite of long shoot Trichoderma, and the preparation method of this insecticide active substance and the application in biological control plant insect thereof.Present inventor finds, containing active ingredient Bemisia tabaci and strawberry spider being had to kill activity in the metabolite of long shoot Trichoderma.This bacterial strain is submitted to China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) preservation on January 14th, 2014 by Wan Lihua bio tech ltd, present invention applicant Shanghai, and preserving number is CGMCC No.8723.
The theme of a first aspect of the present invention is a kind of method preparing insecticide active substance from the secondary metabolite of long shoot Trichoderma, it is characterized in that, comprising:
Step 1: liquid fermentation is carried out to the long shoot Trichoderma that can produce insecticide active substance, obtains the culture fluid containing insecticidal activity metabolite;
Step 2: the culture fluid containing insecticidal activity metabolite obtained slightly is extracted operation, obtains the crude extract of insecticide active substance.
Further, described method also comprises the step 3 of the crude extract of described insecticide active substance being processed further obtained a kind of insecticide, specifically comprise: the various carriers such as the solid phase liquid phase using agricultural chemicals known in the art general and emulsifier dispersion moisture agent, be mixed with the insecticide of any formulation, preferably from missible oil, water dispersible granules, wetting powder, suspending agent, suspension emulsion, any one in effervescent tablet, more preferably, suitably add further and comprise dispersant, wetting agent, emulsifier, bleeding agent, sticker, defoamer, blowing agent, thickener, any one or a few auxiliary agent in stabilizing agent.
In an embodiment of the present invention, described step 1, specifically comprises the long shoot trichoderma strain that can produce insecticide active substance and is inoculated in liquid fermentation medium and cultivates.
Preferably, described long shoot trichoderma strain is China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) preservation, and preserving number is the bacterial strain of CGMCC No.8723.
Preferably, the component of described medium comprises: 5g/L peptone, 30g/L sucrose, 2g/L beef extract, 0.25g/L magnesium sulfate, 0.5g/L potassium dihydrogen phosphate; Wherein, the volume corresponding to concentration of described peptone, sucrose, beef extract, magnesium sulfate and potassium dihydrogen phosphate is the volume of medium.
Preferably, the temperature of described cultivation is 25 ~ 30 DEG C.
Preferably, the mode of described cultivation is: the speed oscillation of 200-500rpm is cultivated 5 ~ 7 days, is preferably the rotating speed of 300rpm.
Further, the fermented product obtained by the above-mentioned medium of this strain fermentation in described step 1 also belongs to the protection domain of this product.
In an embodiment of the present invention, described thick extraction operation is preferably carries out centrifugal filtration by the culture fluid containing insecticidal activity metabolite obtained, separation of supernatant and mycelium, then respectively supernatant and mycelium are carried out to the separation and Extraction of insecticide active substance, merge extract and concentrate, obtaining described crude extract.
Preferably, the separating and extracting process of described supernatant is: by the supernatant of 20BV, after leading to the absorption of HZ806 macroporous absorbent resin with 4BV/h flow velocity, with the alcohol water blend of 2 times of column volumes 20%, 2BV/h flow velocity wash-out removes impurity, use the 50% alcohol water blend 2BV/h wash-out of 2.5BV again, collect eluent.
Preferably, described mycelial separating and extracting process is: filtered after alcohol-pickled 24 hours of 70% by mycelium, collect filtrate, this process repeats twice, then remove alcohol by concentrated for filtrate, then use equal-volume extracting n-butyl alcohol twice, collect and be associated with organic phase extracted solution.
Preferably, described concentrated method is: in 55 DEG C, is concentrated into constant weight in-0.1Mpa vacuum rotary evaporator.
The theme of a second aspect of the present invention is the insecticide active substance that a kind of preparation method described above prepares.
Further, described insecticide active substance can prepare a kind of insecticide further.
The theme of a third aspect of the present invention is a kind of application of insecticide active substance described above, is included in the application that Bemisia tabaci and strawberry spider prevent and treat aspect.
The insecticide active substance contained by secondary metabolite of a kind of long shoot Trichoderma involved in the present invention, to people and animals' avirulence, and meets ecological, environmental protective instructions for use.After tested, insecticide active substance involved in the present invention in use target is strong, the primary extract cream preparation of 8% of preparation is diluted 500 times, reaches more than 90% to the eradicative rate of Bemisia tabaci and strawberry spider.Meanwhile, this primary extract preserves 2 years at normal temperatures, still has virulence.The chemical stability in the present invention with the crude extract of insecticidal activity is strong, and in pH=2.0 ~ 11.0, temperature all has killing action to Bemisia tabaci and strawberry spider under being 10 ~ 100 DEG C of conditions.
Visible, the invention has the advantages that to use described bacterial strain to carry out the control of trialeurodes vaporariorum and strawberry spider safe and reliable, do not produce poisoning, harm humans is unhealthy, free from environmental pollution.
Accompanying drawing explanation
Fig. 1 is in a preferred embodiment of the invention, the described thick conspectus extracting operation.
Embodiment
The invention provides a kind of method preparing insecticide active substance from the secondary metabolite of long shoot Trichoderma, it is characterized in that, comprising:
Step 1: liquid fermentation is carried out to the long shoot Trichoderma that can produce insecticide active substance, obtains the culture fluid containing insecticidal activity metabolite;
Step 2: the culture fluid containing insecticidal activity metabolite obtained slightly is extracted operation, obtains the crude extract of insecticide active substance.
Further, described method also comprises the step 3 of the crude extract of described insecticide active substance being processed further obtained a kind of insecticide, specifically comprise: the various carriers such as the solid phase liquid phase using agricultural chemicals known in the art general and emulsifier dispersion moisture agent, be mixed with the insecticide of any formulation, preferably from missible oil, water dispersible granules, wetting powder, suspending agent, suspension emulsion, any one in effervescent tablet, more preferably, suitably add further and comprise dispersant, wetting agent, emulsifier, bleeding agent, sticker, defoamer, blowing agent, thickener, any one or a few auxiliary agent in stabilizing agent.
In an embodiment of the present invention, described step 1, specifically comprises the long shoot trichoderma strain that can produce insecticide active substance and is inoculated in liquid fermentation medium and cultivates.
Described long shoot trichoderma strain is China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) preservation, and preserving number is the bacterial strain of CGMCC No.8723.
The component of described medium comprises: 5g/L peptone, 30g/L sucrose, 2g/L beef extract, 0.25g/L magnesium sulfate, 0.5g/L potassium dihydrogen phosphate; Wherein, the volume corresponding to concentration of described peptone, sucrose, beef extract, magnesium sulfate and potassium dihydrogen phosphate is the volume of medium.
The temperature of described cultivation is 25 ~ 30 DEG C.
The mode of described cultivation is: the speed oscillation of 200-500rpm is cultivated 5 ~ 7 days, is preferably the rotating speed of 300rpm.
Further, the fermented product obtained by the above-mentioned medium of this strain fermentation in described step 1 also belongs to the protection domain of this product.
In an embodiment of the present invention, described thick extraction operation is preferably carries out centrifugal filtration by the culture fluid containing insecticidal activity metabolite obtained, separation of supernatant and mycelium, then respectively supernatant and mycelium are carried out to the separation and Extraction of insecticide active substance, merge extract and concentrate, obtaining described crude extract.
The separating and extracting process of described supernatant is: by the supernatant of 20BV, after leading to the absorption of HZ806 macroporous absorbent resin with 4BV/h flow velocity, with the alcohol water blend of 2 times of column volumes 20%, 2BV/h flow velocity wash-out removes impurity, use the 50% alcohol water blend 2BV/h wash-out of 2.5BV again, collect eluent.
Described mycelial separating and extracting process is: filtered after alcohol-pickled 24 hours of 70% by mycelium, collect filtrate, this process repeats twice, then removes alcohol by concentrated for filtrate, use equal-volume extracting n-butyl alcohol twice again, collect and be associated with organic phase extracted solution.
Described concentrated method is: in 55 DEG C, is concentrated into constant weight in-0.1Mpa vacuum rotary evaporator.
Present invention also offers the insecticide active substance that a kind of preparation method described above prepares.
Further, described insecticide active substance can prepare a kind of insecticide further.
Present invention also offers a kind of application of insecticide active substance described above, be included in the application that Bemisia tabaci and strawberry spider prevent and treat aspect.
Following examples are only for illustrating to be further expalined technical scheme of the present invention and to illustrate.
Embodiment 1---the preparation of the culture fluid containing insecticidal activity metabolite
(1) first according to microbiology laboratory conventional culture methods, the long shoot Trichoderma bacterium block that picking is stored in 20% glycerine pipe is on a small quantity inoculated in PDA planar surface, cultivates four days for 26 DEG C, as the bacterial classification that shake flask fermentation is cultivated, stand-by;
(2) prepare 20L liquid nutrient medium, be sub-packed in the triangular flask of 100 1000mL, every bottled doses is 200ml, natural pH, 121 DEG C of sterilizing 20min, for subsequent use after cooling,
Wherein, described liquid nutrient medium is 100g peptone, 600g sucrose, 40g beef extract, 5g magnesium sulfate, 10g potassium dihydrogen phosphate, and adding distil water is to 20L;
(3) under aseptic technique, scraping stand-by PDA planar surface Trichoderma spore, is inoculated in sterilizing, is equipped with in the triangular flask of liquid nutrient medium, and be positioned on 30 DEG C of shaking tables and cultivate, rotating speed is 200rpm, cultivates 6 days, stand-by.
The preparation of the crude extract of embodiment 2---insecticide active substance
First the tunning of above-mentioned long shoot trichoderma liquid medium is carried out centrifugal filtration, obtain 16L filtrate and 3L mycelium; After gained filtrate is adsorbed by 800ml HZ806 macroporous resin column with the flow velocity of 3.2L/h, with 2L 20% alcohol water blend, remove impurity with 1.6L/h flow velocity wash-out, then with 2L 50% alcohol water blend, collect active insecticidal components with 1.6L/h flow velocity wash-out; The alcohol-pickled 24hr of gained mycelium 3L 70%, filter, filter residue, with by alcohol-pickled for 3L 70% 24hr, merges twice filtrate, and filtrate is concentrated removes after alcohol with isopyknic extracting n-butyl alcohol 2 times; Finally n-butanol phase active liquid and macroporous absorbent resin 70% ethanol eluate are above merged, be condensed into crude extract 30g.
Embodiment 3---a kind of preparation of missible oil
The crude extract 8g obtained in Example 2, dissolves with 30ml ethanol, adds 40ml 2-butyl acetate and 20ml dimethylbenzene hydrotropy, finally add 10ml emulsifier SP-4099, obtain the missible oil that 100ml content is 8%.
Embodiment 4---a kind of preparation of wetting powder
Crude extract 50g in Example 2, with 2g NNO, 4g sodium lignin sulfonate, 4g neopelex and 40g diatomite co-grinding, obtains the wetting powder that 100g content is 50%.
Embodiment 5---a kind of preparation of water dispersible granules
10 parts of crude extracts in Example 2; with 6 parts of lignin naphthalene sulfonates, 2 parts of ammonium sulfate; 5 parts of mannitol, 5 parts of fatty alcohol sulfate salt, 36 parts of white carbons, 36 parts of kaolin mix, and be ground into Ultramicro-powder, add in fluidized bed pelletizer through micro jet.Contain the aqueous solution of 10% adhesive (water-soluble polyethylene glycol) with 15 parts, in the fluidized bed pelletizer of 50-70 DEG C, carry out granulating and drying, after screening, obtain product.
Embodiment 6---a kind of preparation of suspending agent
Crude extract in Example 2 30 parts, after adding 0.5 part of xanthans, 1 part of polyvinyl alcohol, 3 parts of lignosulfonates, 1 part of lauryl sodium sulfate, 0.5 part of precipitated calcium carbonate, 5 parts of glycerine, 64 parts of deionized water mixing, adopt soybean dietary fiber method sand milling, filter and obtain product.
Embodiment 7---a kind of preparation of effervescent tablet
Crude extract in Example 2 10 parts, add 20 parts of citric acids, 20 parts of sodium bicarbonates, 2 parts pull open powder, 2 parts of BY, 10 parts of talcum powder, 6 parts of polyvinylpyrrolidones, 30 parts of lactose mixing, through pulverizing, granulation, then be drying to obtain product after making definite shape with tablet press machine.
Application Example 8---insecticidal activity is tested
1, experimental design
According to 8% cream preparation that embodiment 3 is prepared, carry out indoor trialeurodes vaporariorum and strawberry spider controlling experiment.Three concentration are established in test altogether, and with clear water in contrast, each process 3 is parallel.
(1) trialeurodes vaporariorum Toxicity Determination
With the Pepper Leaves of card punch cut-off footpath 22mm, then 10s in the medicament of variable concentrations is immersed, (diameter 22mm in the vial that 3ml 2% agar is housed is joined after blade dries, length 78mm), adult is put into wherein, each parallel 30, tampon beyond the Great Wall, measures the survival rate of Bemisia tabaci adults after 48h.
(2) two spotted spider mite Toxicity Determination
Adopt blade infusion process, the From Strawberry Leaves with strawberry spider is immersed 30s in variable concentrations liquid, takes out blade and suck unnecessary liquid with blotting paper.Each parallel guarantee strawberry spider about 30.Finally blade is put into the culture dish of cleaning, temp. and humidity suitable (normal temperature 25 DEG C).Under binocular anatomical lens, check strawberry spider adult death condition after 12h, touch strawberry spider gently with dissecting needle, the motionless person of health is dead.
2, experimental result
Result is respectively as shown in table 1, table 2, and from table 1, table 2, this long shoot Trichoderma all has Bemisia tabaci and strawberry spider extremely strong kills activity, and under this 8% cream preparation dilutes the concentration of 500 times, killing rate is more than 90%.The tunning of the long shoot Trichoderma that this results show is involved in the present invention has that to kill trialeurodes vaporariorum and two spotted spider mite preferably active, may be used for the preparation of microbial pesticide insecticide.
Table 1 8% cream preparation is to the analysis result of Bemisia tabaci
Table 28% cream preparation is to the analysis result of strawberry spider
Be described in detail specific embodiments of the invention above, but it is just as example, the present invention is not restricted to specific embodiment described above.To those skilled in the art, any equivalent modifications that the present invention is carried out and substituting also all among category of the present invention.Therefore, equalization conversion done without departing from the spirit and scope of the invention and amendment, all should contain within the scope of the invention.
Claims (12)
1. from the secondary metabolite of long shoot Trichoderma, prepare a method for insecticide active substance, it is characterized in that, comprising:
Step 1: liquid fermentation is carried out to the long shoot Trichoderma that can produce insecticide active substance, obtains the culture fluid containing insecticidal activity metabolite;
Step 2: the culture fluid containing insecticidal activity metabolite obtained slightly is extracted operation, obtains the crude extract of insecticide active substance.
2. method according to claim 1, it is characterized in that, described method also comprises the step 3 of the crude extract of described insecticide active substance being processed further obtained a kind of insecticide, specifically comprise: the various carriers such as the solid phase liquid phase using agricultural chemicals known in the art general and emulsifier dispersion moisture agent, are mixed with the insecticide of any formulation.
3. method according to claim 2, is characterized in that, described formulation be selected from missible oil, water dispersible granules, wetting powder, suspending agent, suspension emulsion, effervescent tablet any one.
4. method according to claim 1, is characterized in that, described step 1, comprises the long shoot trichoderma strain that can produce insecticide active substance and is inoculated in liquid fermentation medium and cultivates.
5. method according to claim 4, it is characterized in that, described long shoot trichoderma strain is China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) preservation, and preserving number is the bacterial strain of CGMCCNo.8723.
6. method according to claim 4, it is characterized in that, the component of described medium comprises: 5g/L peptone, 30g/L sucrose, 2g/L beef extract, 0.25g/L magnesium sulfate, 0.5g/L potassium dihydrogen phosphate; Wherein, the volume corresponding to concentration of described peptone, sucrose, beef extract, magnesium sulfate and potassium dihydrogen phosphate is the volume of medium; The temperature of described cultivation is 25 ~ 30 DEG C; The mode of described cultivation is: the speed oscillation of 200-500rpm is cultivated 5 ~ 7 days.
7. method according to claim 1, it is characterized in that, described thick extraction operation is that the culture fluid containing insecticidal activity metabolite obtained is carried out centrifugal filtration, separation of supernatant and mycelium, then respectively supernatant and mycelium are carried out to the separation and Extraction of insecticide active substance, merge extract and concentrate, obtaining described crude extract.
8. method according to claim 7, it is characterized in that, the separating and extracting process of described supernatant is: by the supernatant of 20BV, after leading to the absorption of HZ806 macroporous absorbent resin with 4BV/h flow velocity, with the alcohol water blend of 2 times of column volumes 20%, 2BV/h flow velocity wash-out removes impurity, then uses the 50% alcohol water blend 2BV/h wash-out of 2.5BV, collects eluent.
9. method according to claim 7, it is characterized in that, described mycelial separating and extracting process is: filtered after alcohol-pickled 24 hours of 70% by mycelium, collect filtrate, this process repeats twice, then remove alcohol by concentrated for filtrate, then use equal-volume extracting n-butyl alcohol twice, collect and be associated with organic phase extracted solution.
10. method according to claim 7, it is characterized in that, described concentrated method is: in 55 DEG C, is concentrated into constant weight in-0.1Mpa vacuum rotary evaporator.
The insecticide active substance that 11. 1 kinds of preparation methods as described in claim 1-10 any one prepare.
The application of the insecticide active substance that 12. 1 kinds of preparation methods as described in claim 1-10 any one prepare, is included in the application that Bemisia tabaci and strawberry spider prevent and treat aspect.
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| CN110506755A (en) * | 2019-09-09 | 2019-11-29 | 上海万力华生物科技有限公司 | A kind of trichoderma biopesticide effervescent tablet and preparation method thereof |
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| CN110506755A (en) * | 2019-09-09 | 2019-11-29 | 上海万力华生物科技有限公司 | A kind of trichoderma biopesticide effervescent tablet and preparation method thereof |
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