JPH03123713A - Production of insecticide - Google Patents

Abstract

PURPOSE:To obtain an insecticide having improved fungicidal efficiency and high residual insecticidal activity by destroying living spore and living nutritive cell occurring in a culture solution of Bacillus thuringiensis during a specific culture period after removal and separation of water-soluble component followed by pH adjustment. CONSTITUTION:In producing an insecticide having no fear of secondary disaster by eradicating living spore and living nutritive cell existing in a culture solution of Bacillus thuringiensis (BT bacteria), the eradication treatment is carried out in a culture period after 90% release of spore by culture of BT bacteria and within 24 hours from the release and after removal and separation of water- soluble component followed by pH adjustment to 4-7 and to steadily obtain the above-mentioned insecticide having improved fungicidal efficiency, action of gradual sterilization and having extremely high activity by the crystal toxin against residual insecticides, safety, high chemical effects and constant quality.

Description

Detailed Description of the Invention (a) Purpose of the Invention [Field of Industrial Application] The present invention
From a mixture of crystal-like insecticidal protein toxin (hereinafter referred to as crystal toxin) produced by culturing various strains of S. lus thuringiensis (hereinafter referred to as BT bacteria) and spores, etc., while retaining the insecticidal activity of the crystal toxin, spores are generated. Insecticides (hereinafter referred to as BT
It relates to a method for producing agricultural chemicals (referred to as agricultural chemicals) and is widely used in the agricultural chemicals industry and agricultural fields.

[Conventional technology]

Various strains of the BT bacterium produce toxins made of protein, such as summer-shaped, dice-shaped, and amorphous cubes, as they form spores, some of which are selective prephages for the larvae of lepidopteran insects. Others are known to have high insecticidal activity against the larvae of Diptera, such as Aedes and Culex, and those that are selectively toxic only to Coleopteran insects, such as the Colorado-bottomed beetle. It is being

As mentioned above, crystal toxins are extremely selective and do not act on insects that are different from the target species.
Since it is harmless to birds, its use as a selective insecticide has been pursued.

On the other hand, spores, which are living organisms for self-renewal produced through culture, are dormant cells.
It has a strong and durable structure, can withstand environmental conditions (including dry conditions) that are unfavorable to ordinary living things, and can survive for long periods of time. Therefore, when pesticides containing this compound are repeatedly sprayed outdoors, such as in fields, there are concerns that they may accumulate in the soil and disseminate through wind and rain. Strict precautions are being taken as this could lead to

Furthermore, this spore germinates into a vegetative cell, which secretes a water-soluble protein toxin during the process of logarithmic growth, which
It strongly immunochemically reacts with the cereus toxin that causes food poisoning, and clinical testing ■, P, 1559-P, 15
63 (198B)), there is therefore concern that food poisoning may occur during the cooking and preservation process of food with spores attached.

These concerns would disappear if live spores and crystalline toxins were separated to produce a product that does not contain living spores, but both spores and crystalline toxins are about a few micrometers in size,
Not only are they fine, but their surface charge states are similar, making it extremely difficult to physically and electrochemically separate the two, and the process is impractical for industrial use. On the other hand, the above concerns can also be eliminated by manufacturing insecticides that kill spores, but as mentioned earlier, spores have a durable structure and cannot be used for normal killing. Physical processes such as heating, drying, chemical treatment, etc.
Or, it has the strongest resistance among living organisms to chemical sterilization treatment. Therefore, in order to completely kill spores, strict sterilization conditions are required; for example, heating
High temperatures exceeding 0°C are required.

However, when treated under such severe conditions, the crystalline toxin, which is the active insecticidal ingredient, is denatured (heat denaturation of the protein), resulting in a loss of insecticidal efficacy, making it impossible to obtain an effective product.

In order to solve this problem, we applied a slow chemical sterilization treatment to the bacterial cells and spores in the culture solution of BT bacteria containing the crystal toxin, which does not cause the crystal toxin to lose its insecticidal ability. A method for producing an insecticide characterized by combining physical sterilization treatments and simultaneously performing them to kill bacterial cells and spores has been proposed (Japanese Patent Publication No. 51-504).
Publication No. 7).

[Problem that the invention seeks to solve]

The above method is an excellent method for killing bacterial cells and spores and is in practical use, but the crystal toxin obtained by this method can be reduced to a practical concentration by sterilization. (BT pesticides are generally used as a 1.000 to 2.000 times formulation water suspension against diamondback moths), and the residual insecticidal activity tends to be reduced, and when completely killing bacterial cells and spores, However, in some cases, this method has the problem that unless it is used at a considerably high concentration, an insecticide that does not exhibit sufficient insecticidal performance can be obtained.

The present inventors have conducted intensive studies to improve the efficiency of killing bacterial cells and spores in the above method and to establish a manufacturing method that can yield products of excellent quality.

(B) Structure of the Invention [Means for Solving the Problems] In the process of studying to solve the above-mentioned problems, the present inventors discovered that, depending on the culture period of the culture solution to be sterilized, The remaining insecticidal activity changes, the water-soluble components present in the culture solution reduce the sterilization efficiency of vegetative cells and spores, and the insecticidal activity of crystal toxins, and the pH of the culture solution to be sterilized. We discovered that the residual insecticidal activity of crystalline toxins after sterilization changes in response to fluctuations, and by removing water-soluble components from a culture solution cultured for a specific period of time and adjusting the pH, we developed an insecticide that can be put to practical use. The present invention was completed based on the discovery that not only can crystal toxins with insecticidal activity sufficient for the production of insecticidal toxins be consistently obtained, but also crystal toxins with dramatically improved insecticidal activity can be obtained.

That is, the present invention relates to Bacillus thuringiensis (B.
acillus thuringiensis) after 90% of the spores have been released by the culture and 24 hours after the release.
The present invention relates to a method for producing an insecticide, which is carried out after subjecting a culture solution within hours to a step of removing and separating water-soluble components and adjusting the pH to 4 to 7.

○Culture solution of BT bacteria As the culture solution used in the present invention, a BT strain that produces crystal toxins such as Bacillus thuringiensis var. kurstaki, Bacillus thuringiensis var. Examples include general culture solutions obtained by culturing according to the method and conditions.

For example, when Bacillus thuringiensis is cultured using a culture solution consisting of meat extract, peptone, etc. under normal methods and conditions, spores and crystal toxins are formed, and the culture solution that is present during a predetermined culture period, or An aqueous suspension containing crystal toxin and spores obtained by partially purifying or concentrating a culture solution, and a suspension obtained by diluting them with water are used.

More specifically, the culture is carried out in a natural medium rich in nitrogen sources, carbon sources, minerals and vitamins. The production of crystal toxin and bacterial cells depends on the aeration and agitation conditions, and the production amount of both increases when cultured under sufficient aerobic conditions.The culture temperature is preferably about 25 to 30°C. As the carbon source, for example, sucrose, maltose, glucose, fructose, and densitum are used, and as the nitrogen source, for example, corn steep liquor, ammonium sulfate, ammonium chloride, cottonseed flour, yeast extract, soybean flour, casein thawed product are used. In addition, minerals and vitamins can be substituted with dense corn steep liquor yeast extract, and if necessary, inorganic salts and vitamins may be further added.Especially for mass production. If you do,
Deep aeration agitation culture is preferable.

O Setting the culture period The progress from the time spores begin to be released outside the cells until 90% of the spores are released outside the cells is determined by microscopically examining the culture fluid collected aseptically over time using a phase-contrast optical microscope. This makes it easy to track. Spore release means that the crystal toxin and spores formed inside the cells are agitated as the culture progresses and the cell wall of the BT bacterium undergoes self-digestion, and as a result, they are released outside the cells.
Refers to the state of floating in the culture medium.

Spores are elliptical objects with a short axis of 1 to 3 μm x a long axis of 2 to 6 μm, and have light refractive properties, so within the field of view of a phase contrast optical microscope,
They can be distinguished from other granules because they emit a bluish-white light. Crystal toxins and spores can also be distinguished by known staining methods (Fadel A, 5harifet al., J.
Ind. Microbiol, 3+ 227-229
(1988)).

Furthermore, the fact that 90% of the spores were released outside the cells means that the ratio of free spores to the total number of bacteria within the microscopic field (total number of cells containing crystal toxins/spores and free spores) is within the range. You can check it. Although there are some differences depending on the culture conditions, it takes 8 to 24 hours from when spores begin to be released outside the cells until the above state is reached. In the present invention, the culture solution in which 90% of the spores have been released outside the cells is sterilized within 24 hours.

If sterilization is carried out outside the above range, the insecticidal activity of the crystal toxin will be impaired, making it impossible to regularly produce an effective insecticide at a practical concentration, and the residual insecticidal activity will not improve dramatically. .

O Removal and separation of water-soluble components The culture fluid cultured as described above contains components originating from the medium, metabolites excreted outside the bacterial cells by the BT bacteria, or contained within the bacterial cells, and are removed from the culture fluid after autolysis. There are also water-soluble components such as metabolites released into the water, and although the present inventors have not elucidated which of these components they are, it is believed that the presence of these water-soluble components improves the efficiency of sterilization. In order to completely sterilize as it is, that is, in the presence of water-soluble components, the sterilization conditions must be made quite severe.
They found that this ultimately reduces the insecticidal activity of the crystal toxin.

By removing water-soluble components before sterilization, it is now not only possible to produce effective insecticides at practical concentrations, but depending on the degree of removal of water-soluble components, it is now possible to This makes it possible to produce insecticides with insecticidal activity that was previously impossible.

Examples of methods for removing water-soluble components from the culture solution in which spores and crystal toxins have been formed include methods of removing the aqueous solution using conventional centrifugation, filtration, sedimentation, and the like. In particular, when processing a large amount, it is preferable to use a centrifugal separator or a membrane filtration material. In the former case, the amount of water-soluble components removed depends on operating conditions such as centrifugal force (gravitational acceleration), liquid flow interval, and washing interval.
In the latter case, although this will vary depending on the operating conditions such as membrane pore size, pressure, and liquid flow rate, it is preferable to avoid removing too much, which would impede the dispersibility of spores and crystal toxins.
If the dispersibility is inhibited, it is preferable to adjust the dispersibility by adding distilled water, especially a surfactant, or by applying ultrasonic treatment.

If materials with poor dispersibility are sterilized as is, the sterilization efficiency will be inconsistent, which may be due to contact with the sterilizer or poor heat transfer, and especially if complete sterilization is desired, sterilization conditions must be made quite severe. This may reduce the insecticidal activity of the crystal toxin.

The surfactant to be used is preferably an anionic or nonionic surfactant because the surfaces of particles to be dispersed, that is, vegetative cells, spores, and crystalline toxins, are negatively charged. , Sodium polyacrylate type, such as Aron A-20px (manufactured by Toagosei Kagaku Kogyo ■), Sodium dodecylbenzenesulfonate type, such as Perex Nα6 (manufactured by Kao ■), Lebenol WZ (manufactured by Kao ■), Sodium lauryl sulfate type, e.g. Examples of nonionic surfactants include Emar 2F (manufactured by Kao ■), dioctyl sulfosuccinates such as Perex OTP (manufactured by Kao ■), and nonionic surfactants such as polyoxyethylene ethers such as Emulgen 910 (manufactured by Kao ■). can give. The amount used is preferably in the range of 0.01 to 0.50% by weight, particularly preferably 0.05 to 0.20% by weight, based on the culture solution to be sterilized. It is preferable to set the setting according to the operating conditions, etc.

OpH adjustment method In the present invention, the culture solution of BT bacteria is sterilized at pH 4 to 7.
, preferably pH 5-7, particularly preferably pH 5-5.
Since the culture solution generally has a pH of about 8 to 9, the pH is adjusted to 4 to 7 using an acid such as sulfuric acid before sterilization. Also,
During the sterilization process, even if the pH changes due to the process and there is a risk that it may deviate from the above range, it is necessary to add acid or alkali according to the change to maintain the pH within the above range. .

If sterilization is carried out outside the above range, the insecticidal activity of the crystal toxin will be impaired, making it impossible to regularly produce an effective insecticide at a practical concentration, and the residual insecticidal activity will not improve dramatically. .

This effect is also shown when the culture supernatant is removed by centrifugation and resuspended in water.

O sterilization The sterilization in the present invention is carried out to kill vegetative cells and spores, and there are chemical sterilization treatment and physical sterilization treatment methods as described below.
As disclosed in Publication No. 047, merely one type of sterilization treatment can maintain the insecticidal ability of the crystalline toxin.
Since it is difficult to completely kill vegetative cells and spores, it is preferable in the present invention to combine slow chemical sterilization treatment and physical sterilization treatment and perform them simultaneously. It is possible to completely kill cells and spores, and to obtain an industrially extremely useful insecticide with excellent insecticidal ability.

○Chemical sterilization treatment Chemical sterilization treatment methods include formalin, para-toluenesulfone chloramide sodium, para-toluenesulfonic acid dichloramide, benzenesulfonic acid chloramide sodium, azobischloroformamidine, acriflavine, methylene blue benzalkonium chloride, cetyl chloride. N-halides of aliphatic and aromatic dicarboxylic acid imides such as pyridinium, sodium hypochlorite, N-chlorosuccinimide, N-bromosuccinimide, N-chloroglutaric acid imide, and N-chlorobenzoic acid imide This is a method of sterilizing by adding an appropriate amount of a drug such as N-halogendicarboxylic acid imide to the above-mentioned aqueous culture solution.

The amount of the drug added to the culture solution can be easily determined by conducting preliminary tests.

O Physical sterilization treatment The physical sterilization treatment method is a method of sterilizing the above-mentioned aqueous culture solution etc. by heating, ultrasonic waves, radiation, etc.

Industrially advantageous heating methods are the batch heating method, which heats while stirring in a reaction tank, or the continuous flow tube method, which flows into a long reaction tube from one end, heats it midway through, and discharges from the other end. The latter method is particularly useful in industrial implementation.

○ Formulation method The sterilized culture solution can be used as it is, or if necessary, subjected to solid-liquid separation, concentration, and purification, with or without the addition of appropriate auxiliaries, containing crystal toxins and dead spores. It can be made into a suspension-like insecticide. Such an insecticide is preferably formulated into a wettable powder or granule product by a known method such as spray drying or fluidized drying, and further suspended in a solvent.

Although the amount of the active ingredient varies depending on the target insect, it is usually used within a known range, and it can also be used in combination with other insecticides.

○Measurement method for insecticidal activity As a method for quantitatively understanding the insecticidal activity of crystal toxins,
A method was adopted in which the half-lethal concentration was determined through an insecticidal test using live insects, and the residual insecticidal activity was quantitatively measured. That is, the mortality rate of test insects is measured for people using an appropriately diluted sample, the half-lethal concentration is determined from the relationship between the concentration of the sample solution and the mortality rate, and the level of insecticidal activity is compared.

[Effect]

It is unclear why the sterilization treatment of the present invention is effective in improving sterilization efficiency and maintaining the insecticidal activity of crystal toxins, but according to the method of the present invention, the sterilization treatment can be performed more slowly (for example, chemical sterilization). This not only makes it possible to reduce the amount of insecticide used (reducing the amount of insecticide used), but also prevents a decline in the insecticidal activity of the crystal toxin (it also makes it possible to constantly supply insecticides of consistent quality, and is less predictable than with conventional methods). It has the effect of dramatically increasing insecticidal activity to an extent that is impossible to achieve.

〔Example〕

Next, the present invention will be explained in more detail with reference to Examples.

Example 1 Meat extract 1%, peptone 1%, NaCj! 0.5%, p
H7.0 culture raw material solution was heat sterilized at 110°C for 10 minutes, and Bacillus thuringiensis variety kurstakii HD-1 strain (strain stored at the Brandsville Laboratory, United States Department of Agriculture) was added to meat extract and peptone agar. Inoculum that had been statically cultured for 148 hours at 30°C was inoculated onto the slope, and
Shaking culture was performed at C for 5 days. After the 2nd day of culture,
Crystal toxin and spore formation were observed in approximately 50% of the cells.
From the 3rd day, the spores were released outside the cells, and on the 485th day, the spore release rate (method described below) was 90%. The same culture solution corresponded to 15 hours after 90% of the spores were released from the cells. In this culture, the pH of the culture solution is approximately 8. Contain the same amount of this culture solution per unit weight, and have a pH of 1,
The culture solution subjected to 10 types of sterilization treatments such as Prepare the culture solution with sterile water. Transfer this culture solution to a tabletop centrifuge (1
Centrifuge at 4.00 rpm, 4° C. for 110 min), and collect the supernatant by decantation. 10% of the supernatant liquid is replaced with water and returned to the residue in the centrifuge tube for redispersion.

Add several drops of Pellex OTP diluted liquid, which is a surfactant, to this liquid so that the concentration in the liquid is 0.02%,
Dispense 5d of the solid liquid into test tubes, and add 5rnI of the bactericidal agent to this.
A pre-prepared chemical solution with the following conditions was quickly added to the same test tube, subjected to the prescribed heat treatment, cooled to room temperature, and heated to 15.00 O
rpm for 10 minutes, the supernatant was discarded, and 5 d of sterile water was added to the sediment to suspend it, which was repeated twice to remove the bactericidal agent and extracellular soluble toxins. .

(The following is a blank space) Table 1 shows the results of measuring the number of surviving cells/spores (k/d) and residual insecticidal activity of each sample solution obtained in this manner according to the following method. At pH 1, 9, and 11, no mortality was observed and the half-lethal concentration could not be measured even when the sample solution was administered to diamondback moths in an undiluted state with any of the fungicides used. For chlorine-based and oxygen-based disinfectants, high residual insecticidal activity can be achieved by adjusting the pH of the culture solution to be sterilized to between 4 and 7, preferably within the range of 5 to 5.5. is obtained.

Culture fluids are appropriately aseptically collected during various stages of bud culture, and the culture conditions are observed under a phase-contrast optical microscope (1,500x magnification, oil immersion method). Five visual fields were arbitrarily selected per culture solution sample, the spore release rate was determined individually, and the average value was taken as the spore release rate in each culture process.

The spore release rate is expressed as the ratio (%) of the number of free spores to the total number of spores present in the field of view (total number of cells containing crystal toxins and spores in their own bodies and spores released in the culture medium). That's it.

・ Take Xl sample solution 1, dilute it appropriately with sterile water, and add meat extract.
Pour onto a peptone agar plate, culture at 30°C for 48 hours, count the colonies that develop, and calculate the number of surviving cells and spores (ke/d) in the sample.

(Left below) A series of test solutions with 5 to 6 concentration levels was prepared by appropriately diluting the residual JuB1 dilution sample solution with water, and 20
Soak 0d fresh cabbage leaves for 1 minute. After air-drying fresh cabbage leaves, spread them on a large petri dish and release 30 third-instar diamondback moth larvae in each area.After 72 hours, count the number of dead diamondback moths (mortality rate (%)).
Calculate.

This result can be expressed using Finney's graphical method (Finney).
ney.

D.J., (1947) Probit Analysis,
Cambridge University.

Press, Cambridge, 318PP) to determine the half-lethal concentration (concentration of the sample solution in the test solution; ppm).

Example 2 Bacillus thuringiensis variety israelensis HD-522 strain [U.S. Department of Agriculture Brands
[Bacterial strain kept at Bill Research Institute] with 1% meat extract and 0.5 peptone.
%, yeast extract 0.5%, pH 7.0 heat sterilized culture raw material solution 10! inoculated and kept at 30°C, 5 per minute! Culture was carried out with aeration and agitation in anaerobic air. When the culture solution was appropriately aseptically collected during various stages of culture and the culture status was observed using a phase contrast optical microscope, the spore release rate reached 90% after 45 hours of culture.

Cultivation was completed in 60 hours, and the final pH was 8.5. The pH of this cultured solution was adjusted to the level shown in Table 2, sterilized in the same manner as in Example 1, and the number of surviving cells and spores was measured for each solution.

To measure the insecticidal power, each sterilized liquid was diluted with distilled water to create a graded concentration diluted liquid, and each of the two relatives was injected into Culex
20 floating 30 3rd instar larvae of
It was added to the culture solution at 0 d, kept at 27°C, and 2 days later, the number of dead insects was measured, the half-lethal concentration was determined, and the activity retention rate (%) was calculated by comparing it with the value for the culture-completed solution. . The results are shown in Table 2.

As in Example 1, it can be seen that high residual insecticidal activity can be obtained by adjusting the pH of the culture solution to be sterilized to between 4 and 7, preferably within the range of 5 to 5.5.

(Left below) Example 3 Bacillus thuringiensis variety kurstaki HD-1 strain was placed in a Sakalo flask in a C mound (curcose 1%, corn steeple strength -1%, Mn, lppm: p
H7,0) was inoculated in 50m and cultured with shaking at 30°C for 13 days. As in Example 1.2, the spore release rate was tracked, and 90% of the spores were released after 2.5 days of culture. After adjusting the pH of this culture solution to 5.5, the pH was adjusted to 0.8! Hitachi continuous centrifuge rotor (capacity 1ffi)
), and varied the rotation speed (gravitational acceleration: 1,00
0xg to 7,000xg), perform batch centrifugation. After centrifugation, carefully remove the supernatant so as not to include the precipitate, and calculate the removal rate of water-soluble components (concentration ratio: ×1
, ×5, ×10) different concentrated cultures are obtained. In the same method, add a few drops of Emulken 910 diluted solution, which is a surfactant.
Add and mix 5 d of the solution dropwise so that the concentration in the solution is 0.25%, dispense into test tubes, add 5 components of the bactericide used in Example 1 diluted appropriately, After heat treatment under predetermined conditions, the sediment from which the drug was removed was collected in the same manner as in Example 1, suspended in sterile water, and the number of surviving cells/spores and residual insecticidal activity were measured. did. Table 3 shows the minimum value of the drug in the mixture at which the number of viable spores is 0/Id and the residual insecticidal activity of the same method.

As is clear from Table 3, the amount of drug used in terms of culture solids (drug concentration in the mixture/culture solids concentration in the mixture) increases as the removal rate of water-soluble components from the culture solution increases. In addition to being able to reduce this, it has also become possible to dramatically increase the residual insecticidal activity per culture solid content.

(Margin below) Example 4 A culture raw material solution containing 0.5% meat extract, 0.5% peptone, 2% C3L, and 1% glucose was heat sterilized, and Bacillus thuringiensis variety was added to it.

Kurstaki HD-1 strain is inoculated with a seed culture solution that has been precultured for 7 hours, and cultured with aeration at 30°C.

From the early to middle stages of the culture, the pH of the culture solution is adjusted to a range of 6.75±0.25 by adding aqueous ammonia, and after the middle stage, the pH is allowed to rise, and the culture is terminated in 54 hours. As in Example 3, the spore release rate was tracked, and 90% of the spores were released after 38 hours of culture. The pH at the end had reached 8.8.

Add sulfuric acid to this to adjust the pH to 5.5, and
Thickened mud (×5) is obtained by passing through a continuous centrifugal settler at m. Add 0.1 of sodium dodecylbenzenesulfonate to this.
After adding 5% sodium paratoluene sulfone chloramide, add 0°15% and heat at 60°C.
and held for 10 minutes.

The number of surviving cells/spores was 0 cells/d, and the insecticidal activity retention rate determined by the insecticidal power measurement described below was 60%.

The sterilization treatment solution was sequentially diluted with tap water to which 0.03% of Dyne (Takeda Pharmaceutical Co., Ltd.), a sterilizing plate fixing agent, was added to prepare a graded concentration and dilution series.

Cabbage leaf pieces punched into circles with a diameter of 8 cm are immersed in the diluted solution and air-dried. Ten 3rd instar larvae of the diamondback moth are placed on this leaf piece, and the number of dead insects is determined after 3 days. Determine the half-lethal concentration for each level in triplicate, and calculate the activity retention rate (%) by comparing it with the value for the culture-completed solution.

Example 5 After adding Emulgen 910 to 0.12% of centrifugal sedimentation concentrated mud with a pH of 5.5 obtained in the same manner as in Example 4, sodium hypochlorite was added to 0.09% (as available chlorine). ) and continuously fed from one end of the double glass inner tube, 55°C hot water was flowed into the outer tube in parallel flow, and the temperature was raised to 50°C with a piston flow, and at that temperature. Hold for 10 minutes, then
Let it flow continuously.

Number of surviving cells/spores Oke/d, and insecticidal activity retention rate 63
I got %.

Example 6 Bacillus thuringiensis var. israelensis) ID-522 strain was grown in 2xC medium (glucose 2
%, cone steeple force -2%, Mn2ppm: p
H7.0) and cultured with aeration and stirring for 60 hours.

The pH at the end of the culture had reached 8.9. After adding hydrochloric acid to the solution to adjust the pH to 5.5, the supernatant was removed by centrifugation at 10,000 rpm and 1 m to obtain a silt containing crystal toxins and live spores (×10 concentration). Treat with surfactant in the same way.

Next, 0.10% (as available chlorine) of sodium hypochlorite was added, and the temperature was raised to 50°C and held for 10 minutes. According to Example 2, the number of surviving cells and spores and the insecticidal power against Culex mosquito larvae were measured, and the results showed that the number of surviving cells and spores was Oke/Id and the insecticidal activity retention rate was 62%.

(c) Effects of the invention The present invention is effective against the larvae of lepidopteran insects such as the diamondback moth, the cabbage moth, the armyworm moth, and the Japanese armyworm.
Regarding the method of manufacturing insecticides that does not have secondary growth due to viable cells and spores, the culture solution of BT bacteria containing crystalline toxins is subjected to a specific treatment and then sterilized, thereby eliminating the use of chemicals. It has the excellent effect of enabling slower sterilization by significantly reducing the amount of sterilization, and dramatically increasing the residual insecticidal activity after sterilization treatment, greatly contributing to improving the productivity and quality of the insecticide. However, since it is possible to industrially and safely manufacture and provide a safe and highly effective BT pesticide that does not cause secondary disasters due to viable bacteria, it can contribute widely to the agrochemical industry and the agricultural field.

Claims (1)

[Claims] 1. Bacillus thuringiensis
thuringiensis). 90% of the spores were killed by culturing.
After the release and within 24 hours after the release, the culture solution is subjected to a separation process for removing water-soluble components, and the pH is adjusted to 4 to 4.
7. A method for producing an insecticide, characterized in that the method is carried out after adjusting the insecticide.