CN103160449A - Bacillus thuringiensis MB-15 strain and preparation method of its wettable powder - Google Patents
Bacillus thuringiensis MB-15 strain and preparation method of its wettable powder Download PDFInfo
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Abstract
The invention discloses a Bacillus thuringiensis MB-15 strain which has a broad spectrum and high virulence on vegetable lepidoptera pests, and the preservation number of the strain is CGMCC No. 5255. The strain can form a diamond insecticidal crystal protein, contains cry1I, cry1Ac, cry2Ac and vip3Aa genes, and has high-efficiency insecticidal activity on plutella xylostella, Pieris brassicae, beet armyworm, Spodoptera litura, Helicoverpa armigera and other lepidoptera pests. The invention also relates to a preparation method of the wettable powder of strain. The wettable powder comprises the following content of main components: 40.0-50.0% of Bacillus thuringiensis MB-15 strain powder, 5.0-10.0% of a dispersant, 5.0-10.0% of a wetting agent, 0.5-1.0% of a stabilizer, 0.5-1.0% of an ultraviolet light protection agent, and 28.0-49.0% of a carrier. The wettable powder can be used to control a plurality of lepidoptera pests on vegetables, and has the characteristics of broad spectrum, high efficiency, and safety, etc.
Description
Technical field
The invention belongs to the biological prevention field of Agricultural pests.The present invention relates to a strain and lepidoptera pest on vegetables is had the bacillus thuringiensis bacterial strain of the high virulence of wide spectrum, also relate in addition the preparation method to this bacterial strain wettable powder.
Technical background
Bacillus thuringiensis (
Bacillus thuringiensis, be called for short Bt), be the wide gram positive bacterium of a kind of distributed pole, its preparation be at present in the world the microbial pesticide of output maximum (explain sub-ox. the production of sporeine preparation and application [M]. Beijing: agriculture press, 1993.).It is widely used in 9 purpose various insects such as control lepidopteran, Diptera, Coleoptera, Orthoptera (Schnepf E., Crickmore N., Van Rie,
Et al.
Bacillus thuringiensisAnd its pesticidal crystal proteins[J].
Microbiol Mol Biol Rev, 1998,62:775-806.).The main active ingredient of Bt preparation is its parasporal crystal (parasporal crystal protein) that forms in the stable growth phase, claim again insecticidal crystal protein (insecticidal crystal protein, ICP), ICP can account for 20% ~ 30% of culture biomass gross dry weight, molecular weight is (Chattopadhyay A. between 27 ~ 150kDa, Bhatnagar N.B., Bhatnagar R. Bacterial Insecticidal Toxins[J]. Critical Reviews in Microbiology, 2004,30 (1): 33-54.).Find from the beginning of this century, bacillus thuringiensis is the history of existing more than 100 year so far, is widely used aspect the preventing and treating of farm crop and gardening plant insect, injurious forest-insect and sanitary insect pest.But along with the continuous expansion of Bt range of application, Bt is as the defective of sterilant also exposed day by day, and show as: insecticidal spectrum is narrow, and desinsection speed is slow, wherein the generation of resistant insects most serious of all.From last century the mid-80, resistance problem (the William H. M. Insect resistance to the Biological insecticide that all is confirmed in laboratory and field test
Bacillus thuringiensis[J] .Science.1995,229(4709): 193-195), reason is mainly to continue to use single variety and the Bt of sublethal dose and the application of Bt transgenic anti-insect plants, and the selective pressure that insect population is subject to sterilant for a long time causes.Up to the present, under the indoors artificial selection condition, confirm to adhere to separately nearly 20 kinds of insects such as lepidopteran, Coleoptera, Diptera and can produce resistance (Bauer.L.S, 1995. Resistance:a threat to insecticidal crystal proteins of to Bt bacterial strain or toxin
Bacillus thuringiensis[J] .Florida Entomologist, 78 (3), 414-443.).Small cabbage moth is warned people to the high resistance of Bt performance under the natural condition of field, the resistance problem of insect Bt can not be ignored (Tabashnik B E, Cushing N L, Finson N, et al.Field development of resistance to
Bacillus thuringiensisIn diamondback moth (Lepidoptera:Plutellidae) [J].
Journal of Economic Entomology, 1990,83:1671-1676.Sayyed A.H., Gatsi R., Ibiza-Palacios M. S.,
Et al. Common, but Complex, Mode of Resistance of
Plutella xylostellato
Bacillus thuringiensisToxins Cry1Ab and Cry1Ac.
Appled and Environmental Microbiology, 2005,71 (11): 6863 – 6869).Therefore, for fear of the loss that resistant insects causes, seeking new supper toxic strain and preparation is the effective way that addresses this problem, and this biological control to China also has very important meaning.
Until at present, the formulation that bacillus thuringiensis is commonly used comprises aqueous suspension, the oil suspending agent take organic solvent as medium and the wettable powder take the solid-filling agent as medium take water as medium.Although the novel form such as water-dispersible granules and capsule development in recent years is swift and violent, but no matter from product registration quantity or output, wettable powder still occupies critical role in Preparation Forms of Modern Pesticide at present, and because wettable powder and glassware for drinking water have high affinity, can form stable suspension, sprayable using, have be convenient to transport, preserve, use, easy to use, organic solvent-free, non-environmental-pollution and the advantage such as tooling cost is low, so this patent is with the application forms of wettable powder as Bt.
Summary of the invention
One of purpose of the present invention is to provide the new bacterial strain MB-15 of bacillus thuringiensis that a strain has the high virulence of wide spectrum to larvas such as lepidopteran vegetables pest bollworm, beet armyworm, small cabbage moth, prodenia litura and small whities.
Second purpose of the present invention is that short, unstable properties of the lasting period that exists for sporeine preparation in the past, onset wait problem slowly, and a kind of bacillus thuringiensis MB-15 wettable powder and preparation method are provided.
The 3rd purpose of the present invention has been to provide the purposes of bacillus thuringiensis MB-15 on control vegetables lepidoptera pest.
In order to achieve the above object, the present invention adopts following technical scheme to realize:
Bacterial strain MB-15 provided by the invention is bacillus (Bacillus), and bacillus thuringiensis (Bacillus thuringiensis) is called for short Bt MB-15.Being preserved in the Chinese microorganism strain preservation reason person of management committee on September 17th, 2011 understands the common micro-organisms center (be called for short CGMCC, the address is: No. 3, No. 1, North Star West Road, Chaoyang District, BeiJing, China city institute), preserving number is CGMCC № .5255.
Described bacillus thuringiensis CGMCC №. 5255 screening method is the Sodium acetate-antibiotic screening, with the sodium-acetate selected liq culture medium culturing soil sample (200r/min that contains penicillin sodium salt, 30 ℃) 4h, soil supension is centrifugal, is coated with the LB solid plate after getting 65 ℃ of thermal treatments of supernatant, cultivates 3-4d for 30 ℃, the single bacterium colony smear of picking Bt from the flat board, use coomassie brilliant blue staining, find that a strain contains the Bt bacterial strain of rhomboidan form, with its called after MB-15.
Through evaluation, bacillus thuringiensis CGMCC №. 5255 bacterial strains comprise following information: after 30 ℃ of constant temperature culture 72h, can produce rhombus parasporal crystal (see figure 1); SDS-PAGE electrophoretic analysis demonstration, this bacterial strain mainly produces the protein band (see figure 2) of 130.0kDa and 65.0kDa size; Its crystallin begins a small amount of expression at 48h, and 72-96h is that this bacterial strain produces brilliant peak period during this, and after 120h, parasporal crystal begins to reduce gradually; The growth characteristics no significant difference (see figure 3) that the mensuration of this strain growth characteristic is shown this bacterial strain and reference culture HD-1; Genotype identification shows that this bacterial strain contains
Cry1I, cry1Ac, cry2AcWith
Vip3AaFour kinds of genes (seeing Fig. 4, Fig. 5 and table 1).
Table 1 bacterial strain MB-15 killing gene type is identified
Described bacillus thuringiensis CGMCC №. the cultural method of 5255 bacterial strains is: with the single colony inoculation of Bt MB-15 in the LB liquid fermentation medium, fermentation to most of parasporal crystal comes off in 30 ℃, the shaking flask of 200 r/min, be fermented liquid, fermented liquid is put into centrifuge tube, centrifugal (4 ℃, 12 000 r/min, 5 min), abandon supernatant liquor and leave and take precipitation, precipitation is with the sterilized water washing that fully suspends, repeat 2 times, take the weight in wet base that is precipitated, suspend with sterilized water at last and be mixed with 500 mg/mL bacterium liquid, be used for biological assay.
Described bacillus thuringiensis CGMCC №. 5255 biological activity determination method, as follows respectively: as to adopt leaf dipping method to give birth to survey to prodenia litura, small cabbage moth and cabbage caterpillar second instar larvae.Bollworm and beet armyworm second instar larvae are given birth to survey with artificial diet mixed feeding method.Biological assay the results are shown in Table 2.Table 2 result shows, the lethal final concentration LC of bacterial strain Bt MB-15 to bollworm, beet armyworm, small cabbage moth, prodenia litura and cabbage caterpillar second instar larvae 72h
50Be respectively 7.7 * 10
2μ g/mL, 16.01 μ g/mL, 7.61 μ g/mL, 5.28 * 10
4μ g/mL and 22.68 μ g/mL are than the LC of HD-1
50Low, bacterial strain Bt MB-15 shows the insecticidal properties of high virulence wide spectrum to the Vegetables lepidoptera pest.
The median lethal concentration(LC﹠-{50}) LC of table 2 bacillus thuringiensis MB-15 to 5 kinds of vegetables pest 2 instar larvaes
50
The wettable powder that utilizes above-mentioned bacillus thuringiensis MB-15 bacterial strain to produce, its activeconstituents is the mixture of bacillus thuringiensis MB-15 gemma and insecticidal crystal protein.
Component and the content of bacillus thuringiensis MB-15 wettable powder: bacillus thuringiensis MB-15 bacterium powder 40.0 ~ 50.0%; dispersion agent 5.0 ~ 10.0%, wetting agent 5.0 ~ 10.0%, stablizer 0.5 ~ 1.0%; ultraviolet radiation protectant 0.5 ~ 1.0%, carrier 28.0 ~ 49.0%.
Above-mentioned dispersion agent can be sodium lignosulfonate, NNO, HK-2302 or dispersing agent MF; Wetting agent can be Morwet EFW, Sodium dodecylbenzene sulfonate, washing powder or pull open powder; Stablizer can be Xylo-Mucine or xanthan gum; Ultraviolet radiation protectant can be humic acid, whole milk powder, carboxymethyl cellulose or vitamins C; Carrier can be diatomite, light calcium carbonate or white carbon black; The former powder of described bacillus thuringiensis MB-15 is bacillus thuringiensis MB-15 by fermentation after centrifugal concentrating, then the Bt bacterium powder after lyophilize or spraying drying.
The preparation method of above-mentioned bacillus thuringiensis MB-15 wettable powder: Bt bacterium powder and above-mentioned other auxiliary agents is good according to the said ratio weighing, with mortar grind or the pulverize at low temperature airflow machine be mixed pulverize after, both thing of the present invention.
Toxin protein content (130kDa)＞=2.4% of above-mentioned bacillus thuringiensis MB-15 wettable powder, pH value 7.4, the fineness of sieving by 44um is 98.0%, suspensibility reaches 74%, wetting time is 2 ' 34 "; moisture content is 1.9%, and toxicity evaluation is ﹥ 16000 IU/mg, and the every quality index of the Bacillus thuringiensis wettable powder of developing meets GB/T 16000.3-2004 standard.
The application of mentioned microorganism sterilant on the control vegetables pest.
Advantage of the present invention and positively effect: bacillus thuringiensis MB-15 bacterial strain insecticidal spectrum is wide, and the various vegetables lepidoptera pest is had prevention effect preferably; Bacillus thuringiensis MB-15 wettable powder is used for control lepidopteran vegetables pest, has prevention effect good, and the lasting period is long, and nontoxic residue-free is to people, animal and environmental safety.
For the practicality of patent application of the present invention is described, existing check, measuring method and data with patent application sample of the present invention disclose as follows:
The measuring method of suspensibility
Take sample 1.0g (being accurate to 0.002g), directly be placed in the 250 m1 graduated cylinders that fill 50m1 standard hard water, appropriateness is rocked sample is disperseed, and then uses (30 ± 1) ℃ standard hard water to be diluted to scale, covers stopper.Take graduated cylinder bottom as the axle center, graduated cylinder is turned upside down in lmin 30 times (graduated cylinder being inverted and being returned to original position for once, at every turn about 2sec of used time).Open stopper, vertically put into (30 ± 1) ℃ thermostat water bath, standing 30min.After 9/10 (being 225m1) suspension of content being extracted out in 10～15sec with suction pipe, do not shake or stir the sediment in graduated cylinder, be put in baking oven evaporate to dryness after all washing out with standard hard water several times to constant weight.The suspensibility of proprietary preparation of the present invention is greater than 70%.
The measuring method of wetting time
The accurate hard water 100m1 of label taking injects the 250m1 beaker, this beaker is placed in the thermostat water bath of (25 scholar 1) ℃, makes its liquid level concordant with the liquid level in water-bath.Take 1.0g sample (should be representational uniform powder, do not allow agglomerating, caking) with watch-glass, with whole samples from once being poured over equably on the liquid level of this beaker with beaker mouth flush position, but disturbance liquid level excessively not.Clock with stopwatch immediately when adding sample, until sample all wetting till.Write down wetting time (wetting time accurately to second, can ignore by the fine powder film of staying on liquid level), repeat 3 times, get its mean value, as the wetting time of this sample.The wetting time of proprietary preparation of the present invention was less than 180 seconds.
The measuring method of fineness
Take 5g sample (being accurate to 0.1g), be placed in the 250mL beaker, add approximately 80mL tap water, stir with glass stick, make its complete wetting.Testing sieve is immersed in the water, makes the wire cloth complete wetting.With tap water, sample wetting in beaker is diluted to approximately 150mL, stirs, then all pour in wetting standard sieve, rinse beaker with tap water, washing water are also poured in sieve in the lump, until till in beaker, coarse particles moves in sieve fully.Sample on the mild tap water washing screen of deriving with the diameter rubber tubing that is 9-10mm, flow velocity is controlled at 4-5L/min, and rubber tubing end outlet place keeps concordant with the sieve edge for spending.In the process of sieving and washing, keep current to aim at sample on sieve, it is fully washed, flow to always the water of testing sieve limpid transparent till.Again testing sieve is moved in the basin that fills tap water, moves up and down the testing sieve edge and remain on the water surface, be repeated to sieve without material in 2min till.Discard sieving, resistates in sieving first is charged to one jiao in being transferred to the 100mL beaker of constant weight.Standing, in the beaker particles settling to the bottom, most of water that inclines, heating is done the resistates evaporation is near, then is dried to constant weight in baking oven, then takes out beaker and is cooled to room temperature, weighs.Patent application sample of the present invention passes through the fineness of 44um testing sieve greater than 98.0%.
The preparation storage stability is measured
Cold storage detection method: after accurately weighing 5.0g (being accurate to 0.01g) Su Yun gold gemma wettable powder is stored 14d under (0 scholar 1) ℃, observe preparation and have or not caking phenomenon, and its fineness is measured.After the cold storage of patent application preparation of the present invention, without caking, the particle by the 44um testing sieve is greater than 98.0%.
The toxicity evaluation measuring method
With Bt standard substance (the ProductName green 16000IU/mg Bacillus thuringiensis wettable powder of making a living, available from Hubei health novel pesticide technology company limited) and the Bt testing sample dilute successively, select 5 suitable concentration gradients, carry out biological assay take small cabbage moth as the examination worm by immersion method.Be about to the fresh cabbage leaves of in the same size and flood 10s in individual concentration for the treatment of solution, put into after naturally drying to give birth to and survey box, the consistent second instar larvae of every box access 15 head growths, each is processed and repeats 3 times, does blank with sterilized water.The examination worm is placed under (26 ± 1) ℃, 65% relative humidity, 14h illumination condition to be cultivated, and records 72h and respectively processes the dead borer population of larva.Then utilize DPS 7.05 softwares to calculate respectively Bt standard substance LC
50The LC of value and Bt testing sample
50Be worth, be calculated as follows the toxicity evaluation of testing sample:
In formula: X---testing sample toxicity evaluation
S---standard substance LC
50Value
The P---standard substance are tired
Y---testing sample LC
50Value
Description of drawings:
Fig. 1: the opticmicroscope picture of BtMB-15 bacterial strain gemma and crystal, annotate gemma (→) and crystal (←) in figure
Fig. 2: Bt MB-15 bacterial strain crystallin SDS-PAGE collection of illustrative plates
Fig. 3: the growth curve of Bt MB-15 bacterial strain
Fig. 4: BtMB-15 bacterial strain pcr amplification product collection of illustrative plates, wherein:
M:DNA molecular weight standard (8kb, 5kb, 3kb, 2kb, 1kb, 0.75kb, 0.5kb, 0.25kb, 0.1kb)
1,2,3,4,5 and 6 be respectively: Suni, Sun2, Kun2, Kun3, Vip3A and H
2The PCR product of O
Fig. 5: BtMB-15 RFLP identifies collection of illustrative plates, wherein:
M:DNA molecular weight standard (1kb, 900bp, 800bp, 700bp, 600bp, 500bp, 400bp, 300bp, 200bp, 100bp)
1,2,3 and 4 be respectively: Kun2/Pst Ⅰ ﹠amp; Xba I, Kun3/Pst Ⅰ ﹠amp; EcoR I, Sun2/MspI ﹠amp; Hinc II, Vip3A/HindIII ﹠amp; The EcoR I
Embodiment
Following example is further to explanation of the present invention, should not become limitation of the present invention.
In following embodiment, if no special instructions, executing technique means multiplex in example is the conventional means that outside this area, the technician was familiar with.
In following embodiment, described percentage composition is the quality percentage composition if no special instructions.
In following embodiment, the invention technical scheme of described wettable powder is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Separation and the evaluation of embodiment 1, bacillus thuringiensis MB-15 bacterial strain
Bacterial strain of the present invention has separated Bt MB-15 bacterial strain voluntarily by the inventor from soil, soil derives from Hebei province.
Taking the 10g soil sample puts into 50mL sodium-acetate liquid nutrient medium (peptone 0.5g is housed, yeast powder 0.25g, NaCl0.5g, sodium-acetate 1.8g, water 50ml, adjust pH 7.2-7.4,121 ℃, autoclaving 30min) in shaking flask, adding respectively penicillin sodium salt to final concentration is 400ug/mL, shaking table is cultivated (200r/min, 30 ℃) 4h.The earth suspension 10mL that fetches earth after cultivate finishing puts into the aseptic centrifugal 15min of centrifuge tube 3000r/min, gets upper strata turbid solution 1.5mL in 65 ℃ of water-bath water-bath 15min, turbid solution 0.2mL after heat-obtaining is processed, be coated with LB solid medium (peptone 2g, yeast powder 1g, NaCl2g, agar powder 3.2g, water 200ml, adjust pH 7.2-7.4,121 ℃, autoclaving 30min) flat board is put flat board in 30 ℃ of incubators and is cultivated.The bacterial strain smear of 3-4d similar Bt of picking from the flat board is used coomassie brilliant blue staining, in oily Microscopic observation crystal shape and size, finds that a strain produces the bacterial strain of rhomboidan, and called after MB-15(sees Fig. 1).
Above-mentioned bacillus thuringiensis (Bacillus thuringiensis) MB-15, be preserved in Chinese microorganism strain preservation board of trustee reason person on September 17th, 2011 and understand common micro-organisms center (abbreviation CGMCC, the address is: No. 3, No. 1, North Star West Road, Chaoyang District, BeiJing, China city institute), preserving number is CGMCC № .5255.
In the LB liquid nutrient medium, fermentation to most of parasporal crystal comes off in 30 ℃, the shaking flask of 200 r/min, stops cultivating, and namely gets fermented liquid with the single colony inoculation of Bt MB-15.Fermented liquid is put into centrifuge tube, and centrifugal (4 ℃, 12 000 r/min, 5 min) abandon supernatant liquor and leave and take precipitation, precipitation is with the sterilized water washing that fully suspends, repeat 2 times, take the weight in wet base that is precipitated, suspending with sterilized water at last is diluted to different mass concentration for biological assay.
Immersion method is adopted in biological assay to prodenia litura, small cabbage moth and cabbage caterpillar second instar larvae, fresh Eucommia ulmoides Oliv. leaves of the same size or cabbage leaves are immersed each to be processed and takes out after soaking 10s in liquid to be measured, put into to give birth to after drying and survey box, the consistent second instar larvae of every box access 15 head growths, each processing arranges 3 repetitions, each repeats 15 examination worms, be treated to contrast with sterilized water, be placed in (25 ± 1) ℃, cultivate under 65% relative humidity, 14h illumination condition, record 72h and respectively process the dead borer population of larva; Biological assay to bollworm and beet armyworm second instar larvae adds the brilliant plastc ring of 1.5mL spore with artificial diet mixed feeding method with the 15g feed, and after mixing, average mark is loaded in 24 orifice plates, 1 examination worm of every hole access.Each is processed and repeats 3 times, processes feed with sterilized water and compares, and each processing repeats 3 times, and each repeats 24 for the examination insect, is placed in (25 ± 1) ℃ biochemical cultivation case raising and infects, and statistics 72h respectively processes the larva death toll.
The SDS-PAGE of embodiment 3, crystallin analyzes
bacterium colony in the brilliant separation of born of the same parents of oily Microscopic observation, scraping the lawn that takes a morsel fully is dissolved in 100 μ L aqua sterilisas, vibrate resuspended, centrifugal (4 ℃, 12 000 r/min, 5 min), abandon supernatant, after resuspended with 200mL 1M NaCl, centrifugal (4 ℃, 12 000 r/min, 5 min), abandon supernatant, after using again aqua sterilisa resuspended, centrifugal (4 ℃, 12 000 r/min, 5 min), abandon supernatant, precipitation is resuspended in 20 μ L aqua sterilisas, 2 * the sample-loading buffer that adds equal volume, after mixing, 100 ℃ are boiled 5 min, centrifugal (4 ℃, 12 000 r/min, 1 min), get the 20mL supernatant and be used for SDS-PAGE detection (see figure 2).
The bacterial strain MB-15 that the inclined-plane is preserved is streak culture on the LB solid medium, the single colony inoculation of picking Bt in 50mL LB liquid nutrient medium, 30 ℃, 180r/min overnight incubation.Be forwarded in 200mL liquid LB substratum by 1% inoculum size, 30 ℃, 180rpm shaking culture are every 2h sampling and measuring OD
600, survey altogether 24h, draw the growth curve (see figure 3) of bacterial strain, and observe the growth and development state of bacterial strain in culturing process.
The evaluation of embodiment 5, MB-15 strain gene type
The extraction of the total DNA of MB-15 bacterial strain and plasmid DNA is with reference to Narva method (Narva K, Payne J M, Schwab G E, Hickle L A, Galasan T and Sick A J. Novel Bacillus thuringiensis microbes active against nematodes, and genes encoding novel nematodes-active toocin from Bacillus thuringiensis isolates (P). European Patent Office, EP0462721,1991.).Namely take the total DNA of MB-15 bacterial strain as template, carry out pcr amplification with 31 pairs of universal primers, the results are shown in Figure 4, determine that it contains
Vip3A, cry1, cry1IWith
Cry2Genoid.Will
Cry1, vip3AWith
Cry2The PCR product of genoid carries out rflp analysis, the results are shown in Figure 5, determines that it contains
Cry1Ac, vip3Aa, cry2Ac
Bt bacterium powder in embodiment 6, Bacillus thuringiensis wettable powder must be through fermentation, centrifugal, vacuum lyophilization acquisition, and need when being used for the preparation of follow-up preparation to grind or micronizer mill is pulverized with mortar, then cross the 44um normal test sieve, obtain fine powder just available.
Embodiment 7, preparation biological value are the bacillus thuringiensis MB-15 wettable powder of 26546 IU/mg.
Get bacillus thuringiensis MB-15 bacterium powder 50g, sodium lignosulfonate 5g, sodium laurylsulfonate 10g, xanthan gum 1g, skim-milk 1g, white carbon black 33g is after mixing above-mentioned raw materials, grind or be crushed to the uniform powder below 44um in mortar, can obtain bacillus thuringiensis MB-15 wettable powder in micronizer mill.Take standard substance as reference, detecting its biological value is 26546 IU/mg.
Get bacillus thuringiensis MB-15 bacterium powder 45g, sodium lignosulfonate 5g, sodium laurylsulfonate 10g, xanthan gum 1g, skim-milk 1g, white carbon black 38g is after mixing above-mentioned raw materials, grind or be crushed to the uniform powder below 44um in mortar, get final product to get bacillus thuringiensis MB-15 wettable powder in micronizer mill.Take standard substance as reference, detecting its biological value is 19910 IU/mg.
Because the combination of each composition and adopting parameters is too numerous to mention; therefore the various embodiments described above are only for reference; the various embodiments described above can some variations in addition under not departing from the scope of the present invention, therefore should being considered as of comprising of above explanation is exemplary, but not in order to limit the protection domain of the present patent application patent.
Claims (8)
- One bacillus thuringiensis strain ( Bacillus thuringiensis) MB-15, its deposit number at China Committee for Culture Collection of Microorganisms's common micro-organisms center is CGMCC №. 5255.
- 2. bacterial strain according to claim 1, is characterized in that, this bacterial strain has insecticidal activity to larvas such as lepidopteran vegetables pest bollworm, small cabbage moth, small white, beet armyworm and prodenia lituras.
- 3. bacterial strain according to claim 1, is characterized in that, contains through identifying this bacterial strain Cry1I, Cry1Ac, Cry2AcWith Vip3AaFour kinds of gene genes, the crystallin that this bacterial strain produces mainly by molecular weight approximately two kinds of albumen of 130.0kDa and 65.0kDa form.
- 4. bacterial strain according to claim 1, is characterized in that, the tunning of this bacterial strain can be for the preparation of bacillus thuringiensis MB-15 wettable powder.
- 5. bacillus thuringiensis MB-15 wettable powder according to claim 4; its component and weight percent are: bacillus thuringiensis MB-15 bacterium powder 40.0 ~ 50.0%; dispersion agent 5.0 ~ 10.0%; wetting agent 5.0 ~ 10.0%; stablizer 0.5 ~ 1.0%; ultraviolet radiation protectant 0.5 ~ 1.0%, carrier 28.0 ~ 49.0%.
- 6. the dispersion agent of Bacillus thuringiensis wettable powder according to claim 4 is to select from sodium lignosulfonate, NNO, HK-2302 or dispersing agent MF; Wetting agent is from Morwet EFW, Sodium dodecylbenzene sulfonate, washing powder or pulls open powder and select; Stablizer is to select from Xylo-Mucine or xanthan gum; Ultraviolet radiation protectant is to select from humic acid, whole milk powder, carboxymethyl cellulose or vitamins C; Carrier is to select from diatomite, light calcium carbonate or white carbon black; The bacterium powder is the bacillus thuringiensis MB-15 lyophilized powder after centrifugal concentrating or the brilliant mixture of the born of the same parents after spraying drying by fermentation.
- 7. the preparation method of Bacillus thuringiensis wettable powder according to claim 4, it is characterized in that, take described material according to claim 5, after pulverizing with mortar grinding or pulverize at low temperature airflow machine, mix, both got bacillus thuringiensis MB-15 wettable powder.
- 8. the application of bacillus thuringiensis MB-15 wettable powder according to claim 4 on the control vegetables pest.
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| MD4304C1 (en) * | 2013-08-13 | 2015-04-30 | Институт Зоологии Академии Наук Молдовы | Strain of Bacillus thuringiensis subsp. kurstaki bacteria - bioinsecticide for the control of Lymantria genus lepidopterans |
| CN104611277A (en) * | 2015-02-15 | 2015-05-13 | 中国热带农业科学院椰子研究所 | Bacillus thuringiensis for controlling opisina arenosella walkers, serving as palm pests, and application of Bacillus thuringiensis |
| CN105409927A (en) * | 2015-11-17 | 2016-03-23 | 中国烟草总公司郑州烟草研究院 | Bacillus synergist and synergistic biocontrol bacterium agent |
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| CN104611277B (en) * | 2015-02-15 | 2017-07-04 | 中国热带农业科学院椰子研究所 | One plant of preventing and treating palm insect coconut knits bacillus thuringiensis and the application of moth |
| CN105409927A (en) * | 2015-11-17 | 2016-03-23 | 中国烟草总公司郑州烟草研究院 | Bacillus synergist and synergistic biocontrol bacterium agent |
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| CN107760629B (en) * | 2017-11-24 | 2021-02-02 | 四川农业大学 | Bacillus methylotrophicus B18, wettable powder thereof and application thereof |
| CN107760629A (en) * | 2017-11-24 | 2018-03-06 | 四川农业大学 | A kind of Methylotrophic bacillus B18 and its wettable powder and application |
| CN108486008A (en) * | 2018-03-26 | 2018-09-04 | 延边大学 | Dipel YN108, cultural method and its application to the high virulence of lepidoptera pest |
| CN108486008B (en) * | 2018-03-26 | 2021-08-24 | 延边大学 | Bacillus thuringiensis YN108 with high toxicity to lepidoptera pests, and culture method and application thereof |
| CN109169713A (en) * | 2018-09-11 | 2019-01-11 | 天津农学院 | A kind of Bei Laisi bacillus ZSY-1 wettable powder and preparation method and application |
| CN109169713B (en) * | 2018-09-11 | 2021-04-20 | 天津农学院 | Bacillus belgii ZSY-1 wettable powder and preparation method and application thereof |
| CN111315218A (en) * | 2019-08-09 | 2020-06-19 | 北京大北农生物技术有限公司 | Use of insecticidal proteins |
| WO2021026686A1 (en) * | 2019-08-09 | 2021-02-18 | 北京大北农生物技术有限公司 | Use of insecticidal protein |
| WO2021158420A1 (en) | 2020-02-05 | 2021-08-12 | Dow Global Technologies Llc | Bacillus thuringiensis pesticide formulations |
| CN112695001A (en) * | 2021-02-05 | 2021-04-23 | 湖北省生物农药工程研究中心 | Bacillus thuringiensis with high poisoning activity on noctuidae pests and application thereof |
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