JPS5823365B2 - Saikin Seisatsu Yuzai Oyobi Sono Seizou Hohou - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a bacterial insecticide containing as an active ingredient an insecticidal substance obtained from prenatal bacteria having spore defects, and a method for producing the bacterial insecticide.

Conventionally used chemical pesticides are harmful to humans, livestock, and fish, and there is a high risk of serious injury if handled incorrectly.

Moreover, insect pests themselves are developing resistance to chemical insecticides that have been widely used, and their effectiveness is decreasing.

Therefore, in recent years, research has been actively conducted on the use of natural enemies, insect pathogenic bacteria, viruses, etc. in place of conventional chemical insecticides.

As a representative example, in Europe and the United States, microbial pesticides using Bacillus thuringiensis, which are effective against general agricultural and forestry pests, are being sprayed for practical purposes.

In Bacillus thuringiensis, spores and toxin crystals are formed inside the bacterial body, and normally at the end of culture, the cell wall of the bacterial body ruptures naturally and the spores and crystals are released outside the bacterial body.

When the larvae of a Rhinoptera insect ingest this crystal, the larva dies, but it is difficult to separate the spores and the crystal, so preparations containing both are usually used as insecticides.

[Literature base: For example, Bacillus thuringiensis berliner (A, Krieg, Bacillus
huringiensisBerliner, Berl
in (1961)).

However, since these toxins are also toxic to silkworms, there is a drawback that preparations containing spores with reproductive potential cannot be used immediately in sericulture-producing countries such as Japan.

In other words, if a microbial pesticide containing live spores is sprayed as an insecticide, a large amount of bacteria will grow from the spores, which will scatter, killing the silkworms and causing great damage to the silk industry.

Therefore, it is especially desirable in Japan, where the sericulture industry is thriving, to use formulations that do not contain live spores as insecticides.

Moreover, the repeated use of large amounts of certain types of proliferative bacteria as pesticides is likely to disturb the biota in some way in the future (eventually causing pollution).

Therefore, using something that does not have the ability to multiply satisfies the best conditions for a non-polluting pesticide.

For this purpose, it is thought to first perform a sterilization treatment to eliminate the ability to proliferate, and various physical or chemical sterilization treatments are currently being actively attempted.

Among these sterilization methods, an industrial heat treatment sterilization method is desired because of its simple operation.

However, although bacterial cells and spores can be killed by intense heat treatment of Bacillus thuringiensis, this sterilization method also has the drawback of significantly reducing the insecticidal toxin activity (see Table 1).

In addition, chemical sterilization methods using β-propiolactone and hydrogen peroxide have been reported as drugs that can specifically eradicate only viable bacteria and spores without affecting toxins in the bacteria. Japanese Journal of Sericulture, Volume 40, No. 40, 269
~274 pages (1971)], it is difficult to completely kill spores using these sterilization methods, and β-propiolactone is expensive, and hydrogen peroxide is unstable, making it difficult to handle. There are problems, and both have practical difficulties.

Here, the present inventors tried various experiments to improve the above-mentioned drawbacks, and as a result, by treating antepartum Bacillus thuringiensis, which has spore-forming ability, with a mutagenic agent, only the spore-forming ability was lost. They succeeded in creating a spore-deficient strain that retains the ability to produce insecticidal toxins, completing the present invention.

In other words, this spore-deficient strain can be easily and completely killed by mild heat treatment without reducing the activity of the insecticidal toxin, so by using this spore-deficient strain, it is possible to easily produce completely sterilized insecticidal substances. .

The discovery of a technique for obtaining completely sterilized insecticidal substances without impairing the insecticidal toxin activity by only mild heat treatment is an extremely revolutionary breakthrough in industry.

The microorganism used in the present invention may be any spore-deficient spore-deficient strain of spore-deficient spore-deficient bacteria; an example thereof is the well-known Ad73 derived from Bacillus thuringiensis arerestei induced to mutate by 16C1. Bacillus thuringiensis arestee P15A-
1 (Deposited with the Institute of Fine Arts and Science as the Institute of Fine Arts and Science Deposit No. 2458.

) or Bacillus thuringiensis aizawai I4511, which is a known Bacillus thuringiensis aizawai-derived I73 mutated with 16B1 (
It has been deposited with the National Institute of Fine Arts and Technology as the National Institute of Fine Arts and Science Deposit No. 2459.

)and so on. The microorganisms used in the present invention include all spore-deficient bacteria induced by conventional mutagenesis operations or found in natural soil.

The results of comparing the mycological properties of the spore-deficient strain used in the present invention and its parent strain are described below.

Both mutant strains P15A-1 and I4511 retain the characteristics of the species Bacillus thuringiensis, except for the lack of spore-forming ability.

The above-mentioned mutagenesis methods include, for example, methods using ultraviolet irradiation, X-ray irradiation, or chemical agents (eg, nitrosoguanidine, 2-aminopurine, azaserine, acridine orange, diethyl sulfate, or nitrite).

A specific example of the collection of microorganisms used in the present invention is shown in Experimental Example 1 below, and the degree of spore loss is shown in Experimental Example 2.

Experimental example 1 The parent strain (spore-forming strain) was grown in NB medium (meat extract 0
．． 5chi, polypeptone 1%, salt 0.2%, glucose 0
．． 1%, pH 7.0) and cultured with shaking for about 17 hours, 5 TrLl of this culture solution was subcultured to 100 hemp NB medium, and cultured with shaking for 4 hours.

Using a sterile centrifuge tube, dilute the 1001'ILl culture solution with 5.0
00rllIll Centrifuge for 0 minutes and divide the resulting bacterial cells into 10
0.85% saline containing 1nfI nitrosoguanidine 10
ml and shaken for 30 minutes at 30°C.

Next, the suspension is diluted 1,000 times with NB medium and cultured with shaking at 30°C for 4 hours.

10.100% of the culture solution. Diluted 1.000 times,
0.1 ml of the solution is spread on an NB agar medium, left to stand overnight in a constant temperature room at 30°C, and then left to stand at room temperature for 3 to 5 days.

Colonies with strong mixed light are selected, transplanted onto NB agar medium, and left overnight in a constant temperature room at 30°C.

Next, it was planted in 5 m of 7NB medium with platinum wire and incubated for 48 hours for 3
Culture with shaking at 0°C, examine the culture solution under a microscope, select a culture solution that lacks spores and has destroyed cells due to autolysis, take 1.5 - of the culture solution into a test tube, and incubate at 70°C. Heat treatment for 30 minutes, then 1mA! Mix it with an NB agar medium and leave it in a constant temperature room at 30°C for 2 days.

A strain that does not form colonies is considered a spore-deficient strain.

Experimental example 2 (1) Experimental method A parent strain (spore-forming strain) or a mutant strain (spore-deficient strain) was cultured in NB medium at 30°C for 48 hours, and 15 ml of the resulting culture solution was placed in a test tube. Heat treatment is performed for 30 minutes.

Next, the heat-treated solution was spread on an NB agar medium and incubated at 30°C.
Cultivate with

(2) Results The results are shown in Table 2.

(3) Discussion For both mutant strains, the culture solution was heated at 60°C and 65°C for 48 hours.
Heat treatment at ℃ reduces the number of viable bacteria to zero, but in the case of the parent strain (a strain with spore-forming ability), heat treatment at 80℃ and 90℃ reduces the number of viable bacteria, but even with heat treatment at 90℃, the number of viable bacteria decreases to 104 cells/m
1 of viable bacteria remain.

In other words, none of the mutant strains produced spores at all, indicating that they were spore-deficient strains.

Furthermore, as will be shown later, the ability of this mutant strain to produce insecticidal toxins is almost the same as that of the parent strain.

As is clear from the above facts, the use of spore-deficient strains in the production of bacterial insecticides allows the insecticide to be completely sterilized without impairing the activity of the insecticidal toxin by the industrially easy and advantageous heat sterilization method. In addition, since bacteria with low heat resistance are naturally susceptible to other sterilization methods such as fungicides, the technology for producing insecticides using this spore-deficient strain is an extremely innovative technology in industry. It is.

In addition, the spore-deficient strain used in the present invention is extremely susceptible to bacteriolysis, and approximately 90 percent of the bacteria are lysed to death after the production of insecticidal toxin is completed.

Therefore, it can be used as an insecticide that does not contain much viable bacteria without sterilization.

Spores are very stable in soil and can survive for a long time, so when the environment such as soil changes to an environment suitable for spores to germinate and multiply, the number of bacteria increases rapidly.

Therefore, as described above, when insecticides containing spores and insecticidal toxins are used, a large amount of bacteria proliferates, infects and kills silkworms, and causes great damage to the silk industry.

On the other hand, viable bacterial bodies that do not contain spores are extremely unstable in soil and easily disappear.

Therefore, in the case of a spore-deficient strain, even if an insecticide containing the viable bacteria is used as an insecticide, unlike in the case of a useful child strain, there is very little concern that it will cause great damage to the silk industry.

Therefore, in the case of a spore-deficient strain, a preparation containing the viable cells may be used as a fairly safe insecticide.

However, in terms of safety for silkworms, it is preferable to use a sterilized preparation even in the case of spore-deficient strains.

The active ingredient of the bacterial insecticide of the present invention, the "insecticidal substance obtained from spore-deficient productive bacteria", includes free crystalline toxin, live bacterial cells, dead bacterial cells, mixtures thereof, bacterial cells and/or free Concentrated liquids of culture fluids containing insecticidal toxins or their dry powders are included.

The method for producing the bacterial insecticide of the present invention is carried out as follows.

The spore-deficient productive bacteria are cultured in a commonly used medium and under aerobic culture conditions to obtain free crystal toxin, bacterial cells, or a culture containing bacterial cells and/or free crystal toxin.

Furthermore, when living bacteria are to be completely sterilized, normal sterilization treatment may be applied to the living bacteria or a culture containing the living bacteria and/or free crystal toxin.

Examples of the culture medium used in the present invention include natural or synthetic media containing carbon sources, nitrogen sources, inorganic salts, vitamins, etc. as main components.

As a carbon source, for example sucrose, maltose, glucose, fructose, cansucrose, sugar beet molasses or corn molasses are used; as a nitrogen source, for example ammonium sulfate, ammonium chloride, cottonseed flour, soybean flour or casein molasses are used. .

The culture method used in the present invention may be carried out under commonly used aerobic culture conditions, and aerated deep culture is particularly preferable for mass production.

After culturing, remove insecticidal substances (e.g. crystal toxins, etc.) from the culture solution.
Bacterial cells, etc.) may be collected using commonly used methods such as centrifugation and filtration.

In addition, when concentrating or drying the culture solution containing bacterial cells and/or free crystal toxins to form a powder after completion of the culture, a commonly used concentration method or drying method may be used.

However, in this case, it is desirable to carry out the process at a temperature of about 65° C. or lower (G).

The sterilization treatment methods used in the present invention include commonly used treatment methods, such as physical sterilization methods such as heat treatment, ultrasonic treatment, and radiation irradiation, formalin, hydrogen peroxide, sulfites, nitrofuryl acrylamide, and furilfura. Mide,
Examples include chemical sterilization methods using chlorine compounds, β-propiolactone, nitrites, nitrates, surfactants, ethylene oxide, propylene oxide, etc., and biological sterilization methods such as autolysis, phage treatment, or lysozyme treatment.

Among these, heat treatment sterilization methods and chemical sterilization methods are particularly desirable industrially.

As with general pesticides, the active ingredients of insecticides include kaolin, bentonite, talc, diatomaceous earth,
Flour, sugar, bulking agents such as activated carbon, spreading agents, mucilage agents,
It can be used as a powder, granule, tablet, wettable powder, etc. by adding a stabilizer.

In addition, if necessary, other types of insecticides, fungicides, herbicides, plant growth regulators, synergists, attractants, fragrances, plant nutrients, fertilizers, etc. may be added to the extent that they do not inhibit the activity of insecticidal toxins. , can also be used in combination.

The insecticide obtained according to the present invention is effective against, for example, the diamondback moth, the common moth, the common moth, the cabbage butterfly, the armyworm, the fall armyworm, the white armyworm, the killer whale moth, the cypress moth, the Japanese snail moth, and the like.

Insecticides produced using the spore-deficient strain of the present invention can be used safely without fear of breeding bacteria that harm silkworms, and can be used widely in fields unless the insecticide is sprayed directly on mulberry leaves or silkworms. It can be used to exterminate or control pests in fields, fields, forests, etc.

0 Biological test of bacterial insecticide obtained by the present invention Experimental example 3 (Effect on cabbage butterfly and white armyworm.

) 1. Test method 100~100 vacuum-dried bacterial cells produced according to Example 1 were weighed, 10 ml of distilled water was added thereto, and the mixture was thoroughly stirred to prepare a suspension.

Furthermore, distilled water was added to prepare an aqueous solution with a predetermined concentration.

Next, citrus leaf pieces (for cabbage butterfly) and corn leaves (for white cabbage) are soaked in a prescribed chemical solution,
After confirming that it had adhered to the front and back of the leaves, it was air-dried indoors and fed to insects (10 insects tested) at 25°C.

The number of dead insects after a certain period of time was investigated.

3. Discussion The insecticidal activity of the mutant strain is higher than that of the parent strain at the test concentration of 1.
0m9/rILl indicates that sufficient activity is maintained, although it is somewhat inferior.

Experimental example 4 (Effect on silkworms and elysan.

) 1. The test sample culture solution was washed three times with water, suspended in water again, and diluted with distilled water before use.

The control group used only water.

2. Test insects and test method.

0 Silkworm Bombyx mori Samia cynthia ricini
In both cases, artificial feed was used under long-day conditions (16L/8
D) Silkworms with 2nd to 5th instar larvae raised at 25°C and immediately after molting were used.

Five to 10 zero-origin silkworms were placed in a perforated petri dish covered with paper, and an amount of artificial feed to be completely consumed in about 20 hours was prepared, and this feed was immersed in a prescribed drug and fed immediately.

After one day, the old food was discarded and untreated artificial food was given.

Observations were made daily for 3 days (25°C).

3.Result 4.Discussion The mutant strain has no inferiority in insecticidal power compared to the parent strain.

Experimental Example 5 (Effect of Temperature Treatment) ① Experimental Method In order to produce a completely bactericidal insecticide (no spores, no germs), a spore-deficient strain was heat-treated at 60°C for 30 minutes.

The biological test method was the same as in Example 4.

2. Result parent strain: Bacillus thuringiensis arestee Ad
73 to 16C1 Spore-deficient strain 1: Bacillus thuringiensis arestee P15A-1 Spore-deficient strain 2: Bacillus thuringiensis arestee P8934 *: Dilution rate of broth with water as is: 3, discussion Insecticide of spore-deficient strain Toxins can be removed by heat treatment at 60℃ for 30 minutes.
At 1:00 dilution, there is almost no inactivation and viable bacteria are completely eliminated.

Examples will be specifically shown below, but it goes without saying that the present invention is not limited only to the examples.

Example 1 0.5% meat extract in 500ml capacity Sakalokolben, 1
．． Add 100ml of a medium (pH 7.0) consisting of 0% polypeptone, 0.1% glucose, and 0.2% salt, and add Bacillus thuringiensis arestee P15 to it.
A-1 was inoculated and precultured at 30°C for 18 hours under shaking conditions (120°C).

701 volume test fermenter (stirring number 40 rpm)
), 1.0% polypeptone, 0.1% glucose, 0.
Add a medium (pH 7.0) 3013 consisting of 20% salt, and plant 100 ml of the above preculture solution (bacterial cells) at 30°C.
under aerobic culture conditions (451 sterile air per minute pumped into the test fermenter).

301 culture solution in a refrigerated continuous centrifuge at a rotation speed of 9000.
Continuous centrifugation in rpHl.

The obtained precipitate was suspended in 8007711 water or industrial alcohol and centrifuged at a rotation speed of 9000 rl).
III. Centrifuge for 10 minutes.

Repeat this operation three times. The resulting precipitate is dried under vacuum.

Example 2 Example 1 was followed except that the 301 culture solution was heat-treated at 65° C. for 30 minutes.

Example 3 Bacillus thuringiensis arestee P 15
Example 1 is followed except that Bacillus thuringiensis aizawai I4511 is used in place of A-1.

Example 4 Bacillus thuringiensis arestee P15A-
A powder prepared by mixing 25 parts of the freeze-dried powder of 1- with 75 parts of talc is used in an amount of 100 g or more per 10 ares.

Example 5 Bacillus thuringiensis aizawai I4511
25 parts dry powder of culture solution, 2% sodium dodecylbenzenesulfonate, 2% sodium dinaphthylmethane disulfonate
%, a mixture of diatomaceous earth and clay is mixed and ground to make an irrigation agent.

Example 6 Bacillus thuringiensis aizawai I4511
A powder prepared by mixing 25 parts of a dry powder of a culture solution containing sterilized bacterial cells and 75 parts of diatomaceous earth is used, at least 10 parts per 10 ares.

Claims (1)

[Claims] 1. Bacillus thuringiensis with spore deficiency
A bacterial insecticide characterized by containing as an active ingredient an insecticidal substance obtained from Areste P15A-1 or Bacillus thuringiensis aizawai I4511. 2 Bacillus thuringiensis with spore deficiency
A method for producing a bacterial insecticide, which comprises culturing Areste P15A-1 or Bacillus thuringiensis aizawai 14511 and collecting an insecticidal substance without sterilization. 3 Bacillus thuringiensis with spore deficiency
A method for producing a bacterial insecticide, which comprises culturing Areste P15A-1 or Bacillus thuringiensis aizawai I4511, and collecting an insecticidal substance through sterilization treatment.