CN1310230A - Bacillus thuringiensis strain fermentation process and pesticide application - Google Patents
Bacillus thuringiensis strain fermentation process and pesticide application Download PDFInfo
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- CN1310230A CN1310230A CN 01108274 CN01108274A CN1310230A CN 1310230 A CN1310230 A CN 1310230A CN 01108274 CN01108274 CN 01108274 CN 01108274 A CN01108274 A CN 01108274A CN 1310230 A CN1310230 A CN 1310230A
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Abstract
One kind of bacillus thuringiensis is separated, which has high toxic effect on both lepidoptera and hymenoptera insects and serum H7, and contains cry 1Aa, cry 1Ac, cry 1I, cry 2Ab, cry 11 and other insecticidal protein genes, and four crystal protein components of 130-140 Kda, 90 Kda, 70 Kda and 60 Kda. The biological insecticide produced through liquid fermentation process has obvious killing effect to pine moth, house ant, corn boror, rice leaf roller, cabbage caterpillar, etc. and certain killing effect to bollworm.
Description
The present invention relates to the bacillus thuringiensis that can be used as biotic pesticide that a kind of separation screening obtains, bacillus thuringiensis CGMCC NO.0453 (a kind of bacillus thuringiensis particularly, be kept at China Committee for Culture Collection of Microorganisms common micro-organisms center on May 25th, 2000, it abbreviates CGMCC as, and deposit number is CGMCCNO.0453) seed selection, production technique and in the application in biotic pesticide field.
Standard H
7The bacillus thuringiensis strain of serotype exists virulence low, is difficult to the shortcoming of practical application; The bacillus thuringiensis that is used for sterilant on the market mostly is H3 serotype greatly, has only a 130-140Kda insecticidal proteins band, only lepidopterous insects had high virulence, and to hymenopteran---ant class, orthopteran---locustamigratoria avirulence, antagonism small cabbage moth and beet armyworm virulence are poor, and insecticidal spectrum is narrower.And exist fermentation period than shortcomings such as length, its fermentation period was generally about 33-34 hour.
The objective of the invention is to overcome the deficiency of prior art, the desinsection bacillus thuringiensis that a kind of insecticidal spectrum of seed selection is wide, fermentation time is short is designed its zymotechnique and prepares biotic pesticide.
The present invention is achieved through the following technical solutions:
Feature of the present invention comprises Bacillus thuringiensis subsp.aizawai CGMCC NO.0453 strain itself, zymotechnique and in the application in biotic pesticide field.
The bacterial strain that the present invention relates to collects in China Jiangsu Province Yuntai Shan Mountain Japanese red pine bark in May, 1992 and separates in hymenopteran-Japanese hunchbacked ant (Camponotusjaponicus Mayr) the larva corpse of natural death and get, and in May, 2000 in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, its preserving number is CGMCCNO.0453.
One, this bacterial strain has following character
1, morphological specificity
The bacterial strain Gram-reaction is positive, and vegetative cell is shaft-like, the blunt circle in two ends, Dan Sheng (seldom), doublet, or be that unit is linked to be the short chain shape with the doublet, the μ m of thalline size (0.8-1.2) μ m * (2.4-3.6), peritrichous (accompanying drawing 1-2).Strain culturing promptly begins to form gemma and parasporal crystal about 18 hours.The gemma ovalize does not make sporangiocyst expand, and end is given birth to partially.Parasporal crystal rhombus, sphere, irregular shape and crystal are inlayed, and the remarkable lattice of tool (accompanying drawing 3-5).37 ℃ are cultivated 18 hours down is that visible crystal discharges.Growing 45 ℃ of top temperatures, 10 ℃ of minimum temperatures, the most suitable temperature 32-37 ℃ on the broth culture fully.Bacterial strain is not grow on 5.0 and 10.0 the substratum in the pH value, and the appropriate pH value is 6.7-8.Lawn is thin, and smooth surface is glossy, oyster white, and the edge is root shape branch.
2, genetics characteristic
This bacterial strain has the crystallin component (accompanying drawing 6) of 130-140Kda, 90Kda, 70Kda, four kinds of sizes of 60Kda.Contain crylAa, crylAc, crylI, cry2Ab, polytype insecticidal protein gene such as cryll.This bacterial strain belongs to serotype H
7(Bacillus thuringiensis subsp.Aizawai) is identical with the Bacillus thuringiensis Aizawa subspecies.
3, physiological and biochemical property
A, lecithinase (+)
B, urine enzyme (+)
C, saligenin: reaction is unstable
D, sucrose: reaction is unstable
E, seminose (+)
F, fibres sugars (-)
G, proteolysis (++)
H, polychrom (++)
I, starch hydrolysis (+)
J, pigment (-)
K, mycoderm (-)
L, arginine decarboxylase (+)
m、VP(+)
n、MR(+)
The result of above-mentioned test shows that this bacterial strain belongs to Bacillus thuringiensis subsp.aizawai, but with the standard Bacillus thuringiensis subsp.aizawai obvious difference is arranged, and its saligenin, sucrose react obvious inadequately, and the seminose result is opposite.
Two, Bacillus thuringiensis subsp.aizawai CGMCC NO.0453 strain zymotechnique
Bacillus thuringiensis subsp.aizawai CGMCC NO.0453 of the present invention strain can be carried out fermentative production by following zymotechnique.
1, original strain
The supper toxic strain Bacillus thuringiensis subsp.aizawai CGMCC NO.0453 strain of separation screening is as original strain in the Japanese hunchbacked ant larva body of natural death.
2, slant culture
Slant culture based formulas: Semen Maydis powder 2.4%, soybean cake powder 4.8%, fish meal 1.2%, peptone 0.24%, yeast powder 0.12%, CaCO
30.25%, MgSO
47H
2O 0.085%, KH
2PO
40.12%; By each eggplant bottle (200ml) loading amount 60ml packing eggplant bottle, bevel, inclined-plane are put 37 ℃ and are cultivated after one day, as asepsis growth, can use.
The cultivation of slant strains: will burn red transfering loop cooling, from the sandy soil pipe of preservation bacterium, choose the ring of numbers sandy soil by aseptic technique, evenly coat on the bottle inclined plane culture medium of eggplant, putting 30 ℃ cultivated 3-4 days, the visual inspection lawn is plentiful, surperficial oyster white, no plaque, microscopy does not have assorted bacterium, and 90% above bacillus exfoliation can be used for producing.Original strain is inoculated in evenly coats on the bottle inclined plane culture medium of eggplant, put 30 ℃ and cultivated 3-4 days, be to produce and use slant strains;
3, fermentor cultivation
In cultured eggplant bottle slant strains, add 100 milliliters of sterilized waters, make bacteria suspension, change over to then in 500 milliliters of serum bottles, beyond the Great Wall syringe needle or inoculated tube through sterilizing, put in 80 ℃ of water-baths and handled 20 minutes, the gemma suspension is inserted fermentor tank with pressure differential method; Press fermentor tank volumetrical 60% and drop into substratum.Fermentor cultivation based formulas: soybean cake powder 4%, starch 2.4%, corn steep liquor 2%, yeast powder 0.45%, peptone 0.42%, MgSO
40.06%, KH
2PO
40.12%, CaCO
30.2%, that defoamer 0.01%, pH disappear is preceding 8.5, back 7.0 disappears.Feed 118-126 ℃ of high pressure steam, pressure is 0.6-1.0kg/cm
2, keep 30 minutes high pressure steam sterilizations, be cooled to 35 ℃ after, be 7.0-7.5 with the salt acid for adjusting pH value; At 28-35 ℃, tank pressure 0.5kg/cm
2, air flow 1: 0.8-1.2 (v/v) cultivated 28-29 hour under the condition of stirring 180rpm, had brood cell's capsule of 70%--90% to break, discharge crystal with microscopy, can stop fermentation, put jar.
Three, the preparation of biotic pesticide and application
When definite fermented liquid does not have living contaminants, with HCl fermented liquid pH is transferred to 4.5-5.5 rapidly after putting jar, to prevent the degraded of parasporal crystal.Add sanitas NaCl 5% and Sodium Benzoate 0.3% and make suspension agent, get final product packaging final prod.
This refined biometric sterilant adopts the dilution spray method in use, generally is diluted to 100-200 times of stoste, also can increase and decrease in right amount according to different application targets, and usage quantity is with the blade amount of being of complete moistening protected plant.
The present invention separates the bacillus thuringiensis CGMCC NO.0453 strain that obtains in the hymenopteran-larva body of natural death, can produce rhombus, irregular shape, sphere and inlay the parasporal crystal of variforms such as shape, the crystallin component (130-140Kda, 90Kda, 70Kda, 60Kda) that has four kinds of sizes, this bacterium also contains polytype insecticidal protein gene (crylAa, crylAc, crylI, cry2Aa, cryll etc.).Simultaneously lepidopteran, hymenopteran there is high virulence.And fermentation period had only about 28 hours, and it is longer to have overcome the Bt strain fermentation cycle that now has been used to produce sterilant, shortcoming such as insecticidal spectrum is narrower.Have characteristics such as insecticidal spectrum wide (simultaneously lepidopteran, Hymenoptera, orthopteran being had virulence), insecticidal toxicity height, production fermentation period weak point.
Fig. 1 is thalli morphology (shaft-like) figure of bacterial strain of the present invention.
Fig. 2 is thalli morphology (peritrichous) figure of bacterial strain of the present invention.
Fig. 3 is the variform crystal pattern of bacterial strain of the present invention.
Fig. 4 is the rhomboidan and the crystal mosaic map mosaic of bacterial strain of the present invention.
Fig. 5 inlays shape crystal and crystal lattice pattern thereof for bacterial strain of the present invention.
Fig. 6 is the crystallin SDS-PAGE contrast collection of illustrative plates of standard protein and bacterial strain of the present invention.
Following examples describe technical scheme of the present invention in detail.
Embodiment 1
1, slant strains is cultivated
Slant culture based formulas: Semen Maydis powder 2.4%, soybean cake powder 4.8%, fish meal 1.2%, peptone 0.24%, yeast powder 0.12%, CaCO
30.25%, MgSO
47H
2O 0.085%, KH
2PO
40.12%; By each eggplant bottle (200ml) loading amount 60ml packing eggplant bottle, bevel, inclined-plane are put 37 ℃ and are cultivated after one day, as asepsis growth, can use.
The cultivation of slant strains: will burn red transfering loop cooling, from the sandy soil pipe of preservation bacterium, choose the ring of numbers sandy soil by aseptic technique, evenly coat on the bottle inclined plane culture medium of eggplant, putting 30 ℃ cultivated 3-4 days, the visual inspection lawn is plentiful, surperficial oyster white, no plaque, microscopy does not have assorted bacterium, and 90% above bacillus exfoliation can be used for producing.Original strain inoculation (evenly coating) on bottle inclined plane culture medium of eggplant, is put 30 ℃ and cultivated 3-4 days, be to produce and use slant strains;
2, fermentor cultivation
Press fermentor tank volumetrical 60% and drop into substratum.Fermentor cultivation based formulas: soybean cake powder 4%, starch 2.4%, corn steep liquor 2%, yeast powder 0.45%, peptone 0.42%, MgSO
40.06%, KH
2PO
40.12%, CaCO
30.2%, that defoamer 0.01%, pH disappear is preceding 8.5, back 7.0 disappears.Feed 118-126 ℃ of high pressure steam, pressure-controlling is at 0.6-1.0kg/cm
2, keep 30 minutes high pressure steam sterilizations, be cooled to 35 ℃ after, be 7.0-7.5 with the salt acid for adjusting pH value; In cultured eggplant bottle slant strains, add 100 milliliters of sterilized waters, make bacteria suspension, change over to then in 500 milliliters of serum bottles, beyond the Great Wall syringe needle or inoculated tube through sterilizing, put in 70 ℃ of water-baths and handled 15 minutes, the gemma suspension is inserted fermentor tank with pressure differential method; At 31 ℃, tank pressure 0.3kg/cm
2, air flow 1: 0.6 (v/v) stirs under 180 rev/mins the condition and cultivated 32 hours, has brood cell's capsule of 75% to break, discharges crystal with microscopy, stops to ferment, and puts jar.
3, biotic pesticide preparation
With HCl fermented liquid pH is transferred to 4.5-5.5 rapidly after putting jar, to prevent the degraded of parasporal crystal.Add sanitas NaCl 5% and Sodium Benzoate 0.3% and make suspension agent, get final product packaging final prod.
Embodiment 2
1, slant strains is cultivated
Slant culture based formulas: Semen Maydis powder 2.4%, soybean cake powder 4.8%, fish meal 1.2%, peptone 0.24%, yeast powder 0.12%, CaCO
30.25%, MgSO
47H
2O 0.085%, KH
2PO
40.12%; By each eggplant bottle (200ml) loading amount 60ml packing eggplant bottle, bevel, inclined-plane are put 37 ℃ and are cultivated after one day, as asepsis growth, can use.
The cultivation of slant strains: will burn red transfering loop cooling, from the sandy soil pipe of preservation bacterium, choose the ring of numbers sandy soil by aseptic technique, evenly coat on the bottle inclined plane culture medium of eggplant, putting 30 ℃ cultivated 3-4 days, the visual inspection lawn is plentiful, surperficial oyster white, no plaque, microscopy does not have assorted bacterium, and 90% above bacillus exfoliation can be used for producing.Original strain is inoculated in evenly coats on the bottle inclined plane culture medium of eggplant, put 34 ℃ and cultivated 3-4 days, be to produce and use slant strains;
2, fermentor cultivation
Press fermentor tank volumetrical 60% and drop into substratum, fermentor cultivation based formulas: soybean cake powder 4%, starch 2.4%, corn steep liquor 2%, yeast powder 0.45%, peptone 0.42%, MgSO
40.06%, KH
2PO
40.12%, CaCO
30.2%, that defoamer 0.01%, pH disappear is preceding 8.5, back 7.0 disappears.Feed 118-126 ℃ of high pressure steam, pressure is 0.6-1.0kg/cm
2, keep 30 minutes high pressure steam sterilizations, be cooled to 40 ℃ after, be 7.0-7.5 with the salt acid for adjusting pH value; In cultured eggplant bottle slant strains, add 100 milliliters of sterilized waters, make bacteria suspension, change over to then in 500 milliliters of serum bottles, beyond the Great Wall syringe needle or inoculated tube through sterilizing, put in 85 ℃ of water-baths and handled 20 minutes, the gemma suspension is inserted fermentor tank with pressure differential method; At 33 ℃, tank pressure 0.5kg/cm
2, air flow 1: 0.9 (v/v) stirs under 260 rev/mins the condition and cultivated 28 hours, has brood cell's capsule of 95% to break, discharges crystal with microscopy, stops to ferment, and puts jar.
3, biotic pesticide preparation
With HCl fermented liquid pH is transferred to 4.5-5.5 rapidly after putting jar, to prevent the degraded of parasporal crystal.Add sanitas NaCl 5% and Sodium Benzoate 0.3% and make suspension agent, get final product packaging final prod.
Embodiment 3
1, slant strains is cultivated
Slant culture based formulas: Semen Maydis powder 2.4%, soybean cake powder 4.8%, fish meal 1.2%, peptone 0.24%, yeast powder 0.12%, CaCO
30.25%, MgSO
47H
2O 0.085%, KH
2PO
40.12%; By each eggplant bottle (200ml) loading amount 60ml packing eggplant bottle, bevel, inclined-plane are put 37 ℃ and are cultivated after one day, as asepsis growth, can use.
The cultivation of slant strains: will burn red transfering loop cooling, from the sandy soil pipe of preservation bacterium, choose the ring of numbers sandy soil by aseptic technique, evenly coat on the bottle inclined plane culture medium of eggplant, putting 32 ℃ cultivated 3-4 days, the visual inspection lawn is plentiful, surperficial oyster white, no plaque, microscopy does not have assorted bacterium, and 90% above bacillus exfoliation can be used for producing.Original strain is inoculated in evenly coats on the bottle inclined plane culture medium of eggplant, put 34 ℃ and cultivated 3-4 days, be to produce and use slant strains;
2, fermentor cultivation
Press fermentor tank volumetrical 60% and drop into substratum, fermentor cultivation based formulas: soybean cake powder 4%, starch 2.4%, corn steep liquor 2%, yeast powder 0.45%, peptone 0.42%, MgSO
40.06%, KH
2PO
40.12%, CaCO
30.2%, that defoamer 0.01%, pH disappear is preceding 8.5, back 7.0 disappears.Feed high pressure steam, make a jar temperature reach 118-126 ℃, pressure is 0.6-1.0kg/cm
2, keep 30 minutes high pressure steam sterilizations, be cooled to 35 ℃ after, be 7.0-7.5 with the salt acid for adjusting pH value; In cultured eggplant bottle slant strains, add 100 milliliters of sterilized waters, make bacteria suspension, change over to then in 500 milliliters of serum bottles, beyond the Great Wall syringe needle or inoculated tube through sterilizing, put in 85 ℃ of water-baths and handled 20 minutes, the gemma suspension is inserted fermentor tank with pressure differential method; At 35 ℃, tank pressure 0.8kg/cm
2, air flow 1: 1.5 (v/v) stirs under 260 rev/mins the condition and cultivated 26 hours, has brood cell's capsule of 95% to break, discharges crystal with microscopy, stops to ferment, and puts jar.
3, biotic pesticide preparation
With HCl fermented liquid pH is transferred to 4.5-5.5 rapidly after putting jar, to prevent the degraded of parasporal crystal.Add sanitas NaCl 5% and Sodium Benzoate 0.3% and make suspension agent, get final product packaging final prod.
Embodiment 4:
Bacillus thuringiensis subsp.aizawai CGMCC NO.0453 strain preparation is with 1: 100 times of dilution; be sprayed onto protected plant surface; with the complete moistening amount of being; observe the result of use of this sterilant poisoning dendrolimus spectabilis, cypress caterpillar, Pyrausta nubilalis (Hubern)., cabbage caterpillar, small cabbage moth, Japanese hunchbacked ant; observe the insect mortality ratio of using after 48 hours; reference standard Bacillus thuringiensis subsp.aizawai reference culture sterilant the results are shown in Table one
Table one (48 hours, mortality ratio %)
| Bacillus thuringiensis CGMCC NO.0453 strain | The Bacillus thuringiensis subsp.aizawai reference culture | |||||||
| Concentration | Stoste | 10 -1 | ?10 -2 | ?CK | Stoste | 10 -1 | ?10 -2 | ?CK |
| Dendrolimus spectabilis | 96.5 | ?86 | ?51 | ?2 | ?34.5 | ?24 | ?6 | ?1 |
| The cypress caterpillar | 86 | ?68 | ?55 | ?1 | ?74.5 | ?62 | ?48.5 | ?2 |
| Pyrausta nubilalis (Hubern). | 89 | ?84.5 | ?61 | ?1 | ?30.5 | ?18.5 | ?4 | ?2 |
| Cabbage caterpillar | 100 | ?93 | ?73 | ?0 | ?86.5 | ?73 | ?45 | ?0 |
| Small cabbage moth | 87 | ?83.5 | ?68 | ?0 | ?47.5 | ?31.5 | ?10 | ?1 |
| The hunchbacked ant of Japan | 95.2 | ?87.5 | ?73 | ?1 | ?68.5 | ?56 | ?12 | ?1 |
Embodiment 5
Bacillus thuringiensis subsp.aizawai CGMCC NO.0453 strain preparation was with times dilution in 1: 100,1: 200; be sprayed onto protected plant surface; with the complete moistening amount of being; observe the result of use of this sterilant poisoning small cabbage moth; observe the insect mortality ratio of using after 2 days, 4 days, 6 days; contrast 5% chloro-cyanide pyrethrin insecticide agent, the results are shown in Table two:
Table two: the test of pesticide effectiveness result of bacillus thuringiensis CGMCC NO.0453 strain emulsion control small cabbage moth
| Medicament | Extension rate | Average prevention effect (mortality ratio %) | ||
| 2 days | 4 days | 6 days | ||
| Bacillus thuringiensis CGMCC NO.0453 strain leavened prod | 100 | ?71.66 | ?81.76 | ?84.32 |
| Bacillus thuringiensis CGMCC NO.0453 strain leavened prod | 200 | ?63.71 | ?70.93 | ?74.25 |
| 5% Cypermethrin | 1000 | ?43.36 | ?59.93 | ?63.68 |
Embodiment 6
Bacillus thuringiensis subsp.aizawai CGMCC NO.0453 strain preparation was with times dilution in 1: 200,1: 400; be sprayed onto protected plant surface; with the complete moistening amount of being; observe the result of use of this sterilant poisoning dendrolimus spectabilis; observe the insect mortality ratio of using after 2 days, 3 days, 4 days, the results are shown in Table three: table three: bacillus thuringiensis CGMCC NO.0453 strain emulsion control dendrolimus spectabilis test-results
| Medicament | Average prevention effect (mortality ratio %) | ||
| 2 days | 3 days | 4 days | |
| Dilute 200 times | ????37 | ????85.6 | ????88.3 |
| Dilute 400 times | ????32.5 | ????77.8 | ????79.6 |
| Contrast 0.1% washing liquid | ????0 | ????1.8 | ????1.8 |
Embodiment 7
Bacillus thuringiensis subsp.aizawai CGMCC NO.0453 strain preparation dilutes with the honey proportioning with 1: 100 times, put in the culture dish of 30 tame ants and a make-up, observe this sterilant and kill the result of use of tame ant, with the positive contrast of Bt bacterium C005 strain, the negative contrast of clear water, observe the mortality ratio of using after 3 days, the results are shown in Table four.Table four: bacillus thuringiensis CGMCC NO.0453 strain emulsion is killed tame ant test-results
| Bacterial strain | Mortality ratio % |
| CGMCC NO.0453 strain | ????88.5 |
| Bt bacterium C005 strain | ????10 |
| ????CK | ????3 |
Embodiment 8
Clip 1cm
2The bright leaf of Semen Maydis of size, draw 15 microlitre Bacillus thuringiensis subsp.aizawai CGMCC NO.0453 strain preparations respectively on each blade, after drying naturally, be put in the little vial, throw in a dolly locust 2-3 instar larvae in each vial, after treating that larva is eaten up blade, move and put into diameter and be 10cm, highly be the foster worm tube of 40cm that each tube is thrown in 30 of larvas, with Bt bacterium C005 strain, the positive contrast of pseudomonas, the negative contrast of clear water was observed mortality ratio after 7 days, the results are shown in Table five.Table five: bacillus thuringiensis CGMCC NO.0453 strain emulsion poisoning dolly locust test-results
| Bacterial strain | Mortality ratio % |
| CGMCC NO.0453 strain | ????94.5 |
| Bt bacterium C005 strain | ????14 |
| Pseudomonas | ????32 |
| ????CK | ????9.4 |
Claims (4)
1, a kind of Bacillus thuringiensis subsp.aizawai (Bacillus thuringiensissubsp.aizawai serotype H
7) CGMCC NO.0453.
2, Bacillus thuringiensis subsp.aizawai CGMCC NO.0453 according to claim 1, this bacterial strain has following feature: (1) this bacterial strain belongs to serotype H
7, according to 14 groups of biochemical reactions and other H of international microorganism NK regulation
7Serological type strain is compared, and only Whitfield's ointment, sucrose react unstable, and the N.F,USP MANNITOL result is opposite, and other 11 group reaction results are identical; (2) this bacterial strain has the crystallin component of 130-140Kda, 90Kda, 70Kda, four kinds of sizes of 60Kda; (3) this bacterium contains crylAa, crylAc, crylI, cry2Ab, polytype insecticidal protein gene such as cryll; (4) have rhombus, irregular shape, sphere and inlay the parasporal crystal of variforms such as shape.
3, the zymotechnique of the described Bacillus thuringiensis subsp.aizawai CGMCC of claim 1 NO.0453 strain, its feature mainly comprises following step: (1) is evenly coated original strain on the bottle inclined plane culture medium of eggplant, put 30-34 ℃ and cultivated 3-4 days, be to produce and use slant strains; (2) in cultured eggplant bottle slant strains, add 100 milliliters of sterilized waters, make bacteria suspension, change over to then in 500 milliliters of serum bottles, beyond the Great Wall syringe needle or inoculated tube through sterilizing, put in the 70-85 ℃ of water-bath and handled 15-20 minute, the gemma suspension is inserted fermentor tank with pressure differential method; (3) press fermentor tank volumetrical 60% and drop into substratum, feed high pressure steam and make a jar temperature reach 118-126 ℃, pressure is 0.6-1.0kg/cm
2, keep 30 minutes high pressure steam sterilizations, be cooled to 33-40 ℃ after, be 7.0-7.5 with the salt acid for adjusting pH value; (4) at 31-35 ℃, tank pressure 0.3-0.8kg/cm
2, air flow 1: 0.6-1.5 (v/v) stirs under the 180-260 rev/min of condition and cultivated 26-32 hour, has brood cell's capsule of 70%-90% to break, discharge crystal with microscopy, can stop fermentation, puts jar; (5) bottle inclined plane culture medium of eggplant consists of Semen Maydis powder 2.4%, soybean cake powder 4.8%, fish meal 1.2%, peptone 0.24%, yeast powder 0.12%, CaCO
30.25%, MgSO
47H
2O 0.085%, KH
2PO
40.12%; (6) fermentor cultivation based formulas: soybean cake powder 4%, starch 2.4%, corn steep liquor 2%, yeast powder 0.45%, peptone 0.42%, MgSO
40.06%, KH
2PO
40.12%, CaCO
30.2%, defoamer 0.01%, pH is 8.5 before the froth breaking, pH is 7.0 behind the froth breaking.
4, the described Bacillus thuringiensis subsp.aizawai CGMCCNO.0453 of claim 1 is as the application of the main sterilization component of biological pesticide on poisoning lepidopteran, Hymenoptera, orthopteran.
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| CN101347129B (en) * | 2008-09-01 | 2011-12-14 | 佛山市正典生物技术有限公司 | Bacillus thuringiensis suspending agent for killing mosquito |
| CN102732444A (en) * | 2012-02-09 | 2012-10-17 | 河北省农林科学院植物保护研究所 | Bacillus pumilus for controlling cottonbollblight and microorganism bacterium agent thereof |
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| CN103525834A (en) * | 2013-04-25 | 2014-01-22 | 湖南农业大学 | Crylac gene and application thereof |
| CN111742937A (en) * | 2015-01-16 | 2020-10-09 | 瓦伦特生物科学有限责任公司 | Bacillus thuringiensis and bacillus thuringiensis subspecies silvicaricus combined preparation |
| CN107427013A (en) * | 2015-01-16 | 2017-12-01 | 瓦伦特生物科学有限责任公司 | Cooperate with bacillus thuringiensis and B. thuringiensis subspecies insecticide mixtures |
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| CN106431598A (en) * | 2016-07-23 | 2017-02-22 | 成都金昌旺农业科技有限公司 | Preparation method of amino acid biological phytoalexin |
| 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 |
| CN108575456A (en) * | 2018-03-30 | 2018-09-28 | 叶虹 | The biological control method of corn borer |
| CN108359673A (en) * | 2018-05-08 | 2018-08-03 | 江苏省农业科学院 | A kind of Bt cry11 genes, coding albumen and its application efficiently killing edible mushroom eye fungus gnat |
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