CN1055310C - Supper toxic strain YBT-1520 of thuricin brood cell and its zymosis process and products - Google Patents

Abstract

The invention relates to the selection and breeding of Bacillus thuringiensis and its fermentation process and product development. It is characterized in that a Bacillus thuringiensis VBT-1520 strain with specific and high toxicity to cotton bollworm is isolated, its flagellar antigen serotype is H3ab, and the insecticidal crystal protein gene copy number is cry1A(c)>cryIA( a)>cryIA(b), the fragment of HindⅢ where the N-terminal of the cryIA(c) gene is located is 7.0kb, and the nucleotide sequence of the cryIA(c) gene has four nucleotide differences from the typical cryIA(c). The liquid fermentation process is used to produce biological insecticides, and the control effect on cotton bollworm is over 95%, and it also has good effects on rice leaf curler and corn leafworm.

Description

Bacillus thuringiensis hypervirulence strain YBT-1520 and its fermentation process and products

The present invention relates to a highly virulent Bacillus thuringiensis strain YBT-1520 (a Bacillus thuringiensis preserved in CCTCC, preservation number M94067, preservation date: October 4, 1994) used for biological insecticide obtained by separation and screening Breeding, its production process and the field of biopesticide technology.

At present, the resistance of cotton bollworms to chemical pesticides is increasing day by day. Most chemical pesticides have poor control effects on cotton bollworms, especially resistant cotton bollworms, so that cotton bollworms are rampant in some areas. In the past ten years, many inventors at home and abroad have devoted themselves to screening binary or ternary pesticides and compounding them into pesticide mixtures. Although they have played a certain role in the control of cotton bollworms, the impact of chemical pesticides on the ecological environment and insect natural enemies It goes without saying. Therefore, the development of biological pesticides to control certain harmful insects, especially the selection of strains specific to cotton bollworm and their production preparations, is an urgent problem to be solved in the comprehensive management of agricultural pests.

Biopesticides have the outstanding advantages of not polluting the environment, being safe for humans and animals, insects are not easy to develop drug resistance, and simple production technology. Bacillus thuringiensis insecticide is currently the most important biopesticide, mainly used to control Lepidoptera pests.

Currently the strains used in the world are all HD-1 or similar strains, and its flagellar antigen serotype is H3ab. The HD-1 strain was isolated from the raised pink bollworm by Dulmage in 1970. It was 20-212 times more virulent than the commercial production strain at that time, thus creating a new starting point for Bacillus thuringiensis insecticides. Abbott and Sandoz's Dipel and Javelin product strains are HD-1 and NRD-12 respectively, and the laboratory shake flask fermentation broth has a toxicity titer of about 2000IU/ul to cotton bollworm. There were no detectable differences between the two strains except for the production of thuringeenin by the latter. Different strains of Bacillus thuringiensis are mainly reflected by the differences in their flagellar antigens. The current commercialized strains mainly belong to serotype H3ab, but there are still differences in virulence between strains of the same serotype. Therefore, the identification of a Bacillus thuringiensis It must reflect the number, type and combination of its insecticidal genes. Both HD-1 and NRD-12 contain cryIA(a), cryIA(b), cryIA(c), cryIIA and cryIIB insecticidal crystal protein genes, the first three genes The HindIII fragments at the N-terminus are 4.5kb, 5.3kb and 6.6kb respectively. The copy numbers of cryIA(a) and cryIA(c) genes are the same, but both are lower than those of cryIA(b). 130kDa and 65kDa crystal proteins can be detected in SDS-PAGE electrophoresis, which are the products of three cryId genes respectively (see Science Press, 1990, Bacillus thuringiensis, edited by Yu Ziniu)

The YBT-1520 bacterial strain of the present invention has specific high toxicity to cotton bollworm, belongs to H3ab with HD-1, and compared with HD-1, it produces the same type of crystal protein and contains insecticidal crystal protein gene, However, the location of these genes, the ratio of gene copy number, and the nucleotide sequence of the genes all have obvious differences, especially their overall virulence is higher than that of HD-1 and NRD-12.

The purpose of the present invention is to overcome the deficiencies of the prior art, select and breed a bacillus thuringiensis strain with specificity and high toxicity to pests such as cotton bollworm, design its fermentation process and prepare biological insecticide.

The present invention is realized through the following technical solutions:

The features of the invention include a separation and screening method for bacillus thuringiensis YBT-1520, a liquid submerged fermentation process and preparation of liquid and powder products.

1. Separation and screening method

Acetate selective medium (abbreviated as BPA medium) is used to separate from the soil, and the larvae of Plutella xylostella xylostella are used as test insects for toxicity determination, and the bacterial strain of the present invention is obtained by screening.

1. BPA medium formula: 5 grams of beef extract, 10 grams of peptone, 34 grams of sodium acetate, 1000 ml of distilled water, pH 7.2-7.4.

2. Separation method

Weigh 1 gram of soil sample in BPA medium, shake it fully, put it in a shaker at 30°C for 4 hours, take it out and heat it in a water bath at 75-80°C for 10-15 minutes, after standing still, suck 0.5ml and place it on a BPA plate Spread evenly on the medium, place it upside down in an incubator at 30°C for 24 hours, select 3 to 5 colonies similar to Bacillus thuringiensis and inoculate it on the BPA slant, culture at 30°C for more than 72 hours, check under the microscope with phenol-fuchsin staining, and Isolates with parasporal crystals were identified as Bacillus thuringiensis.

3. Toxicity Assay

3.1 Primary Screening

Bacillus thuringiensis isolates were inoculated onto beef extract peptone medium (beef extract 0.5%, peptone 1.0%, NaCl 0.5%, agar 2.0%, pH7.2) on the slope (18 × 180mm), cultivated at 30°C for more than 72 hours, Add 10ml of sterile water to wash the bacterial lawn, break it into a uniform bacterial suspension as the stock solution, soak the cabbage leaves with a 1:10 dilution of the stock solution, take it out and dry it, put it in an infection bottle, and put 10 third-instar diamondback moth larvae into each bottle , each strain was repeated 3 times, and sterile water was used as a blank control, and the results were checked after 48 hours of infection at 25°C.

3.2 Re-screening

The strains with a corrected mortality rate of more than 90% in the primary screening were re-screened, and the original solution was diluted 1:1000 for the re-screening, and the strain HD-1 was used as a reference standard, and the method was the same as that of the primary screening.

4. Strain culture, subculture and preservation

Cultivate on the slant of beef extract peptone medium at 30°C. It can be stored at 4°C for a short period of time. It can be stored in a frozen tube or a sand tube for a long time. As the original strain, the production strain needs to be transferred from the original strain and inoculated in beef Cream the peptone medium on the slants.

5. Biological and genetic characteristics of YBT-1520 strain

Biological characteristics: the bacteria are straight rod-shaped, the vegetative body is chain-like or solitary, 3-5 microns long, 1-1.2 microns wide, Gram staining is positive, the spores are cylindrical or nearly elliptical, partial terminal, spores The capsule does not expand, and the colony is round on the beef extract peptone medium, with smooth edges and a waxy lawn. Oxyphobia, grow in beef peptone medium containing 7% NaCl, produce acid in ribose, glucose, fructose, maltose and soluble starch medium, methyl red reaction is positive, large parasporal crystals are produced in the late stage of spore growth, parasporal crystals It is diamond-shaped, square, irregular, etc., with different sizes. In the fermentation culture, it can grow vigorously in the medium with various agricultural and sideline products as raw materials, produce a large number of parasporal crystals, and the toxicity titer to insects can reach 3000IU/ul. The heat-treated spores are used to inoculate the seed tank, and the logarithmic growth phase bacteria are used to inoculate the fermenter tank. The growth delay period of the bacteria is short and the synchronization rate is high.

Genetic characteristics: Bacillus thuringiensis YBT-1520 is a highly virulent insecticidal isolate isolated in our laboratory. Its flagellar antigen serotype is H3ab, and its parasporal crystals are composed of 130kDa and 65kDa crystal proteins.

The characteristics of the insecticidal crystal protein gene of this strain are quite different from those of typical H3ab strains. The identification by polymerase chain reaction (PCR) indicated that the strain contained five insecticidal crystal protein genes of cryIA(a), cryIA(b), cryIA(c), cryIIA and cryIIB. This is the same as the typical strain HD-1, but the copy numbers of the first three types of genes are different in this strain. PCR and Southern hybridization showed that the strain had a high copy number of the cryIA(c) gene, followed by a cryIA(a) gene, and a very low copy number of the cryIA(b) gene. The order of copy number is: cryIA(c)>cryIA(a)>cryIA(b), but in HD-1 and NRD12, the copy number of cryIA(b) gene is the highest, and the copy number of cryIA(a) and cryIA(c) same. Southern hybridization also showed that the HindIII fragment at the N-terminal of the cryIA(c) gene was 7.0kb, and the cryIA(b) gene fragment was not displayed, which further indicated that the copy number of the gene was low.

There are four nucleotide differences between the nucleotide sequence of the cryIA (c) gene and the typical cryIA (c) gene sequence (Adang, M.J., etal 1985, Gene.36:289-300), that is, at 1365 , 1368, 1389 and 1407 positions are C, T, G, C, respectively, instead of A, G, T, T.

The strain has high virulence to cotton bollworm (Heliothis armigera) and diamondback moth (Plutella xylostella), and is more virulent than HD-1 and NRD-12.

2. Production process of Bacillus thuringiensis YBT-1520

The fermentation process of YBT-1520 strain as a biopesticide is divided into two parts: liquid submerged fermentation and formulation:

The liquid submerged fermentation process (see Figure 1) and preparation process are as follows:

(1) Original strain

Bacillus thuringiensis YBT-1520 strain with high virulence and stable performance preserved in the microbial pesticide room of Huazhong Agricultural University in frozen tube or sand tube was used as the original strain.

(2) Inclined bacteria

Slant medium formula: beef extract 0.5%, peptone 1.0%, NaCl 0.5%, agar 2.0%, pH7.2. Divide the medium into test tubes or eggplant bottles, sterilize with damp heat (15 psi, 121°C) for 30 minutes, and place them on an inclined plane when the temperature drops to about 50°C. If the temperature is too high and there is a lot of condensed water, the slope can be incubated at 37°C for one day, and it can be used only if it grows sterile.

Cultivation of slant strains: Inoculate the original strains on the slant medium of the test tube, and cultivate them at 30°C for 24 hours for activation. Then transfer from the inclined plane of the test tube to the inclined plane of the eggplant bottle, cultivate at 30°C for 72 hours, observe with the naked eye that the bacterial lawn is plump, the surface is milky white, there is no phage plaque, there is no miscellaneous bacteria in the microscopic examination, and more than 90% of the spores can be used for production. The slant strains for production should be stored in the refrigerator for no more than one week.

(3) Seed tank cultivation

Seed tank medium formula: bean cake powder 3.0-5.0%, corn flour 1.0-5.0%, fish meal 0.3-2.0%, yeast powder 0.1-2.0%, inorganic salt 0.2-1.8%, foam enemy 0.01-0.03%, all raw materials are It needs to pass through an 80 mesh sieve, and the pH after sterilization is 7.0-7.2. 60-80% of the volume of the seed tank is put into the medium, and then the high-pressure steam is directly passed into the fermenter to make the temperature reach 121°C, the pressure is 15 psi, maintain for 30 minutes, cool to 30-35°C, and insert Bacterial suspension made from activated slant strains. The pH value is 8.5 before sterilization and 7.0-7.2 after sterilization.

The medium of the seed tank can also adopt the following formula: bean cake powder 3.5-4.5%, corn flour 1.0-3.0%, fish meal 0.8-1.6%, yeast powder 0.1-0.5%, inorganic salt 0.2-0.8%, bubble enemy 0.01-0.03 %, all raw materials need to pass through an 80-mesh sieve, and the pH after sterilization is 7.0-7.2;

Seed tank inoculation: Add sterile water to the cultured eggplant bottle slant to make a bacterial suspension containing 0.2 to 100 million viable cells per milliliter, transfer it into a 500 milliliter serum bottle, and plug it with sterilized Needle or inoculation tube, and tightly tied with a rope, placed in an 80°C water bath for 15 to 20 minutes to kill free phages and activate spores. The heat-treated spore suspension is connected to the seed tank by the differential pressure method. The pH value of the seed tank culture medium was 7.0 at the time of inoculation.

Seed tank cultivation: at a temperature of 30-32° C., a tank pressure of 0.3-0.5 kg/cm2, and a ventilation rate of 1:0.6-1:0.8 and stirring at 200-250 rpm. Cultivate for 6-12 hours. Observed under a microscope that all the spores have germinated and grown into vegetative bodies. The shape of the bacteria is normal and there is no contamination by bacteria.

(4) Fermentation tank culture

Fermentation tank medium formula: bean cake powder 3.0-5.0%, corn flour 1.0-5.0%, fish meal 0.3-2.0%, yeast powder 0.1-2.0%, inorganic salt 0.2-1.8%, foam enemy 0.01-0.03%, all raw materials are Need to pass through 80 mesh sieve, the pH before sterilization is 8.5, and the pH after sterilization is 7.0-7.2.

The culture medium of the fermenter can also adopt the following formula: soybean cake powder 3.5-4.5%, corn flour 1.0-3.0%, fish meal 0.8-1.6%, yeast powder 0.1-0.5%, inorganic salt 0.2-0.8%, bubble enemy 0.01-0.03 %, all raw materials need to pass through an 80-mesh sieve, the pH before sterilization is 8.5, and the pH after sterilization is 7.0-7.2.

Fermentation culture: 60-70% of the volume of the fermenter is put into the medium, the temperature of the fermenter reaches 121°C through high-pressure steam, the pressure is 15 psi, and the sterilization is maintained for 30 minutes. After cooling to 30-33°C, press 1-10% of the feeding volume is transferred into the seed solution. Cultivate for 24-48 hours at a temperature of 28-32°C, a tank pressure of 0.3-0.8 kg/cm2, an air volume of 1:0.6-1:1.2 (V/V), and stirring at 200-250 rpm. Once 40% of the spores have ruptured, they are ready for canning.

Fermentation control: The pH value of this medium is adjusted to about 8.5 before sterilization, and the pH value after sterilization is 7.0-7.2, so that the germination rate of spores reaches the highest. When the bacteria began to grow, the pH value dropped to 5.8-6.0, and then rose back to about 8.5 when the tank was released. Aeration: In the early stage of fermentation, the number of bacteria is low and the oxygen consumption is low; when entering the logarithmic phase, the oxygen consumption increases, and the ventilation flow rate is generally required to reach 1:1.0 to 1:1.2 (referring to the ratio of the volume of the fermentation medium to the air per minute) volume ratio). When the spores begin to form, although the metabolic activity of the bacteria decreases, oxygen has an important impact on the maturation of the spores and the formation of parasporal crystals. Therefore, a large amount of ventilation should be maintained in the late fermentation period. Temperature: check the temperature value at any time, control The temperature variation is 30±2°C.

(5) post-treatment of fermented product

The fermented liquid is acidified and concentrated in an appropriate amount, and then additives are added to make a liquid product or spray-dried to make a powder.

Liquefaction of fermentation broth: first adjust the pH value of the fermentation broth to 5.0-6.0 with HCl, and then properly concentrate according to the level of the fermentation broth. The fermented liquid is concentrated by 0.5-1 times by using a fluid continuous centrifuge to make the toxicity titer reach the required level, and then 5-10% NaCl and 0.3% sodium benzoate preservatives and other auxiliary agents are added.

Fermentation powder formulation: After the fermented liquid is concentrated to a certain concentration, add an appropriate amount of filler (such as diatomaceous earth or calcium carbonate) and wetting agent (1-0.3% agricultural milk 100), and put it into the high tank on the top of the spray-dried algae tower middle. When the inlet temperature reaches 140-150°C, start spraying, control the speed of slurry flowing into the centrifugal disc, and keep the temperature of the middle layer of the spray drying tower at 60-65°C, so that the water content of the dried preparation is below 4%.

(6) Product quality inspection

The quality inspection of fermentation intermediate products and products was carried out according to the bioassay method established in my country using Plutella xylostella larvae as test insects. Using the standard product [CS3ab-1991, virulence titer 18000IU/mg (international unit/mg)] prepared by this laboratory, the standard product and the sample to be tested were serially diluted, and the infection solution of different concentrations and the artificial diet of diamondback moth Mix and make infected feed, infect third instar diamondback moth larvae, check the larval mortality rate under different concentrations, use statistical method to calculate the _LC50 value of test sample and standard product, calculate the virulence potency of test sample by the following formula:

(7) Product packaging

The preparation is 1 kg in each small package, and the products are divided into several types according to the different active ingredients contained.

Liquid type I: The toxicity potency is 2000IU/ul

Liquid Type II: The toxicity potency is 4000IU/ul

Liquid Type III: The toxicity potency is 8000IU/ul

Powder type I: The toxicity potency is 8000IU/mg

Powder type II: The toxicity potency is 16000IU/mg

Powder type III: The toxicity potency is 32000IU/mg

The dosage forms and specifications of Bacillus thuringiensis are mentioned above, but not limited thereto.

Three, positive results of the present invention

The YBT-1520 name insecticide (commodity name is called for short Mianfeng insecticide) that is made with the present invention and its embodiments selects the powder product control second generation cotton bollworm for use, can reach control purpose, when using 200 times dilutions, control Effect can reach more than 95%; 400 times of dilution can reach more than 90%, and 400 times of Dipel powder (commercial bacillus thuringiensis preparation produced by U.S. Abbott Company) and 1000 times of 20% methomyl control effect are equivalent (see Table 1) . Because the cotton bollworm is resistant to chemical pesticides, the invention and its products have high use value and popularization value in the control of the cotton bollworm when the chemical control is out of control.

The field control effects of YBT-1520 powder (Mianfeng insecticide) against rice leaf roller and corn borer are shown in Table 2 and

Table 1 Field control effect of YBT-1520 powder (II type) against the third generation cotton bollworm potion Dilution factor Average control effect (%) one day four days six days Mianfeng Insecticide Powder Type II 200 98.9 99.2 99.4 400 90.0 92.8 90.3 600 82.8 86.0 70.3 Dipel powder (16000IU/mg) 400 76.0 86.2 88.0 20% methomyl 1000 90.2 86.6 87.4

Table 2 Field control effect of YBT-1520 powder (Type II) on rice leaf roller potion Dilution factor Control effect (%) one day three days five days YBT-1520 powder 1000 44.8 71.3 71.7 2000 25.9 57.5 64.0 Dipel powder (16000IU/mg) 1000 36.5 62.6 73.2 50% Methamidophos 1000 70.9 76.6 77.3

As shown in table 3, it can be seen from table 2 and table 3 that 1000 times of Mianfeng insecticide powder is equivalent to the Dipel powder control effect of rice leaf roller and the same dilution multiple, for corn borer 30g/kg granule and 50 The control effect of %1605 granule was equivalent.

Table 3 Field efficacy of YBT-1520 powder in controlling the first-generation corn borer potion Granule concentration Average decline rate (%) bored stem bored hole Mianfeng Insecticide Powder Type II 30g/kg15g/kg7.5g/kg 80.3 82.968.4 63.660.6 59.8 50% 1605 10ml/kg 75.7 78.4

Example:

1. Inclined bacteria

(1) Strain source: the highly virulent strain YBT-1520 screened by our laboratory.

(2) Preparation of inclined plane

Culture medium formula: 5 grams of beef extract, 10 grams of peptone, 5 grams of NaCl, 20 grams of agar, 1000 milliliters of water, pH 7.2, the culture medium is subpackaged, and the subpackage test tube (18×180 mm) has a capacity of 10 ml, and is plugged with a cotton plug; Each eggplant bottle (200ml) has a capacity of 60ml, plugged with a cotton plug, wrapped with a layer of gauze inside and a layer of kraft paper outside, and sterilized by moist heat (15 psi, 121°C) for 30 minutes until the temperature dropped to 50 When it is around ℃, it is placed on a slope, and the slope is placed at 37°C for one day, and it can be used only if it grows aseptically.

(3) Cultivation of Inclined Strains

Cool the red-hot inoculation loop, dip it in a little sterile water (not too much, so as not to drip into the sand tube to wet the sand), pick a ring of sand from the sand tube according to the aseptic operation, and spread it evenly on the slope of the test tube On the medium, culture at 30°C for 24 hours for activation, then transfer from the slope of the test tube to the slope of the eggplant bottle, remove the kraft paper, wrap only a layer of gauze (to facilitate ventilation), and culture at 30°C for 72 hours. Milky white bag, no phage plaque, microscopic examination without miscellaneous bacteria, more than 90% of spores can be used for production. The slant of each eggplant bottle contains more than 30 billion viable spores.

2. Seed jar culture

Seed tank medium formula: bean cake powder 3.2%, corn flour 2.0%, fish meal 0.8%, yeast powder 0.16%, inorganic salt 0.3%, foam enemy 0.01%, pH adjusted to 8.5. All raw materials need to pass through 80 mesh sieve. Drop into the culture medium by 65～80% of the volume of the seed tank, then directly feed the high-pressure steam into the fermenter to make the temperature reach 121° C., 15 pounds per square inch, and keep for 30 minutes. Throughout the sterilization process, continuous agitation is necessary to avoid aggregation and precipitation of solid matter in the medium. The stirring speed may be maintained as long as no precipitation of solid matter occurs. During the cooling process, a certain tank pressure must be ensured to avoid bacterial contamination.

Seed tank inoculation: Add sterile water to the cultured eggplant bottle slant strain to make a bacterial suspension containing 0.2 to 100 million viable cells per ml, then transfer it to a 500 ml serum bottle, plug it and sterilize it Take the needle or inoculation tube, and tie it tightly with a rope, and keep it in an 80°C water bath for 15 to 20 minutes to kill free phages and activate spores. The heat-treated spore suspension is introduced into the seed tank by the differential pressure method. When inoculating, the temperature of the seed tank is 30-35°C, and the pH value is 7.0-7.2.

Seed tank cultivation: Cultivate for 6-12 hours at a temperature of 30-35°C, tank pressure of 0.3-0.5 kg/cm2, ventilation rate of 1:0.6-1:0.8, and stirring at 200-250 rpm. Observed under a microscope that all the spores have germinated and grown into vegetative bodies. The shape of the bacteria is normal and there is no contamination by bacteria.

3. Fermenter culture

Fermentation tank medium formula: bean cake powder 3.2%, corn flour 2.0%, fish meal 0.8%, yeast powder 0.16%, inorganic salt 0.3%, foam enemy 0.01%, pH adjusted to 8.5. All raw materials need to pass through 80 mesh sieve.

Fermentation culture: 60-70% of the volume of the fermenter is put into the medium, and high-pressure steam is introduced to make the temperature of the fermenter reach 121°C, the pressure is 15 psi, maintain for 30 minutes, cool to 30-33°C, and the pH is 7.0～7.2. Transfer the seed liquid according to 1-10% of the feeding volume; at a temperature of 28-35°C, a tank pressure of 0.3-0.8 kg/cm2, a ventilation rate of 1:0.6-1.2 (V/V), stirring at 200-250rpm and normally open conditions Cultivate for 24-48 hours under the microscope, and after checking with a microscope, 20-40% of the spore capsules are ruptured, and then they can be put into a tank.

4. Post-treatment of fermented product

When the titer of the fermented liquid is stable at 2000-3500IU/ml and there is no contamination by bacteria, it can be acidified, concentrated in an appropriate amount, added with additives to make a liquid product and spray-dried to make a powder.

A. To adjust the pH, the pH of the fermentation broth should be adjusted to 5.0-6.0 with HCl immediately after putting into the tank to prevent the degradation of the parasporal crystals.

B. Concentrate. According to the level of fermentation broth (virulence titer), the fermentation broth is concentrated by 0.5 to 1 times with a fluid continuous centrifuge, so that the toxicity titer reaches the required level.

C. Liquefaction. Add preservative (NaCl 5% and sodium benzoate 0.3%) and wetting agent (2% Nongru 100) to the fermented liquid that has been acidified and concentrated, and then the finished product can be packaged.

D. Powdering agent. Add appropriate amount of filler (diatomaceous earth) and wetting agent (3% Nongru 100) to the acidified and concentrated fermented liquid, and pour it into the high tank at the top of the spray drying tower. The temperature of the middle layer of the spray drying tower is kept at 60-65° C., so that the water content of the dried preparation is below 4%.

5. Product quality inspection

The quality inspection of fermentation intermediates and products was carried out according to the bioassay method established in my country using the third instar larvae of diamondback moth as test insects. The standard product is CS3ab-1991 prepared by our laboratory with a potency of 18000IU/mg (International Unit/mg).

Claims (5)

1. supper toxic strain of producing biotic pesticide has cryIA (a) | and cryIA (b) and cryIA (c) insecticidal crystalline gene is characterized in that, described bacterial strain is YBT-1520, a kind of Tribactur Bacillus thuringiensis, CCTCC NO.M94067

It comprises:

The copy number of three insecticidal crystalline genes of A, this bacterial strain is followed successively by: cryIA (c)＞cryIA (a)＞cryIA (b);

The HindIII segment at the terminal place of B, cryIA (c) gene N-is 7.0kb;

The nucleotide sequence of C, cryIA (c) gene and typical cryIA (c) gene nucleotide series have the difference of four Nucleotide, promptly 1365,1368, are respectively C, T, G, C on 1389 and 1407 nucleotide positions, rather than A, G, T, T.

2. liquid fermentation process that utilizes Tribactur as biotic pesticide, the used bacterial strain of this method is the supper toxic strain YBT-1520 of thuricin brood cell that separation screening obtains, a kind of Tribactur, CCTCC, NO.M94067, its feature may further comprise the steps:

A, original strain is inoculated on the test tube slant substratum, cultivates for 30 ℃ and activated in 24 hours, transfer then, cultivated 72 hours for 30 ℃, be to produce and use slant strains in eggplant bottle inclined-plane;

B, in cultured eggplant bottle slant strains, add sterilized water, make every milliliter of bacteria suspension that contains 0.2～1.0 ℃ of hundred million viable cell, change over to then in the 500ml serum bottle, syringe needle or the inoculated tube of bacterium beyond the Great Wall went out, bottle put be incubated 15～20 minutes in 80 ℃ of water-baths bacteria suspension is heat-treated, with pressure differential method this bacteria suspension is inserted seeding tank, this jar medium pH is 7.0～7.2 during inoculation;

C, press seeding tank volumetrical 60～80% and drop into substratum, high pressure steam sterilization (15 pounds/square inch, 121 ℃) was kept 30 minutes, be cooled to 30～35 ℃ after, insert the bacteria suspension that activated inclined-plane lawn is made;

D, press fermentor tank volumetrical 60～70% and drop into substratum, high pressure steam sterilization is promptly at 121 ℃, pressure is 15 pounds/square inch, stirs under 200～250rpm condition, keeps sterilization in 30 minutes, after being cooled to 30～33 ℃, move into seed liquor by 1～10% of the volume that feeds intake;

E, seed tank culture are at 30～32 ℃ of temperature, tank pressure 0.3～0.5kg/cm ², air flow 1: 0.6～1: 0.8 (V/V) stirs under 200～250rpm condition and cultivated 6～12 hours;

F, fermentor cultivation are at 28～32 ℃ of temperature, tank pressure 0.3～0.8kg/cm ², air flow 1: 0.6～1: 1.2 (V/V) stirs under the 200.250rpm condition and cultivated 24～48 hours, have brood cell's capsule of 20～40% to break with microscopy after, just can put jar;

G, seed tank culture based formulas are: soybean cake powder 3.0～5.0%, Semen Maydis powder 1.0～5.0%, fish meal 0.3～2.0%, yeast powder 0.1～2.0%, inorganic salt 0.2～1.8%, bubble enemy 0.01～0.03%, all raw materials all needed 80 mesh sieves, pH is 8.5 before the sterilization, and sterilization back pH is 7.0～7.2;

H, fermentor cultivation based formulas are: soybean cake powder 3.0～5.0%, Semen Maydis powder 1.0～5.0%, fish meal 0.3～2.0%, yeast powder 0.1～2.0%, inorganic salt 0.2～1.8%, bubble enemy 0.01～0.03%, all raw materials all needed 80 mesh sieves, pH is 8.5 before the sterilization, and sterilization back pH is 7.0～7.2;

3. method according to claim 2 is characterized in that:

A, seed tank culture based formulas are: soybean cake powder 3.5～4.5%, Semen Maydis powder 1.0～3.0%, fish meal 0.8～1.6%, yeast powder 0.1～0.5%, inorganic salt 0.2～0.8%, bubble enemy 0.01～0.03%, all raw materials all needed 80 mesh sieves, pH is 8.5 before the sterilization, and sterilization back pH is 7.0～7.2;

B, fermentor cultivation based formulas are: soybean cake powder 3.5～4.5%, Semen Maydis powder 1.0～3.0%, fish meal 0.8～1.6%, yeast powder 0.1～0.5%, inorganic salt 0.2～0.8%, bubble enemy 0.01～0.03%, all raw materials all needed 80 mesh sieves, pH is 8.5 before the sterilization, and sterilization back pH is 7.0～7.2;

4. method according to claim 2, when it is characterized in that ferment tank enters logarithmic phase, its air flow is: 1: 1.0～1: 1.2 (V/V).

5. the production method of biotic pesticide, the used bacterial strain of this method belongs to the supper toxic strain YBT-1520 of thuricin brood cell that separation screening obtains, a kind of Tribactur, CCTCC NO.M94067, and, it is characterized in that according to the state of the art of claim 2 and 3:

A, fermented liquid pH is transferred to 5.0～6.0 with HCl, according to the fermented liquid level, adopt whizzer that fermented liquid is concentrated 0.5～1 times, make toxicity evaluation reach desired level, add 5～10%NaCl, 0.3% Sodium Benzoate and 1～3% farming breast 100 then, through check, be the liquid dosage form biotic pesticide after the packing.

B, in concentrated broth, add an amount of diatomite or lime carbonate and 1～3% farming breast 100, make toxicity evaluation reach desired level, spray-dried operation is controlled at below 4% the preparation water content, through check, is the pulvis biotic pesticide after the packing.

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