JPH0310681A - Bacillus-thuringlensis variant and insecticide - Google Patents

Abstract

NEW MATERIAL:To provide a Bacillus.thuringlensis variety crustaki variant losing a spore-forming ability and a part or all of plasmid holding a gene coding an insecticidal protein having an insecticidal ability against Lepidoptera insects. USE:An insecticide. PREPARATION:For example, the spores of Bacillus.thuringlensis variety crustaki HD-1 are suspended in a phosphate buffer solution, heated at 70 deg.C for 20min, mixed with a mutagen such as ethylmethane sulfonic acid, shaken and subsequently centrifuged. The prepared precipitates are cultured in a slant medium, and a sporedefective variant is selected from the cultured products and subjected to a plasmid-removing treatment to provide a MA31-0 strain. The prepared MA31-0 strain and the original strain not subjected to the mutation treatment are shaken and cultured in test tubes, respectively, and subsequently mixed with each other, and the mixture is coated on an agar medium and cultured at 30 deg.C for 3 days to provide a variant strain MA31-2.

Description

DETAILED DESCRIPTION OF THE INVENTION 1.Objective of the invention ``Field of industrial application'' The present invention is directed to a Bacillus thuringiensis variety that exhibits an extremely excellent insecticidal effect on the larvae of lepidopteran insects (cabbage butterflies, diamondback moths, etc.). By deleting the plasmids possessed by K. kurstaki that each contain genes encoding different insecticidal proteins, the plasmids remained in the bacterial body.
express only genes encoding arbitrary insecticidal proteins,
The active ingredient is a Bacillus thuringiensis variety kurstaki mutant strain that specifically produces the insecticidal protein expressed therein and does not form spores, and an insecticidal protein obtained by culturing the mutant strain. It relates to insecticides that are widely used in the agrochemical industry and agricultural field.

``Prior Art'' Insecticidal proteins, which are intracellular inclusion bodies formed during the sporulation stage of various Bacillus thuringiensis species, are called crystal toxins and are known to be toxic to the larvae of many insects. In addition, the insecticidal activity of the crystal toxin has been
For example, the crystalline toxins produced by subspecies Kurstaki and Aizawai are used for Lepidoptera and Diptera insects, and the crystalline toxin produced by Israelensis is used for Diptera insects. Furthermore, the crystalline toxin produced by Tenebrionis is known to be effective against Coleoptera. Therefore, conventionally, these crystal toxins have been widely used as insecticides, and in this case, strains that are considered to be most effective against the pests to be controlled are selected.

``Problem to be solved by the invention'' An insecticide containing crystalline toxin as an active ingredient, which is produced using a culture solution of Bacillus thuringiensis in the spore-forming stage, is
For reasons described below, relatively high manufacturing costs are required.

That is, since crystalline toxins are produced only during the spore-forming stage of the producing bacteria, fermentation in industrial fermenters takes a considerable amount of time, resulting in poor production efficiency.

Furthermore, if a normal Bacillus thuringiensis culture solution is used as the base for an insecticide, it contains many spores that can germinate, so after the insecticide is sprayed in nature, The spores contained within germinate,
There is a risk of promoting the growth of bacteria and causing chemical damage to silkworms. Therefore, from the standpoint of protecting the sericulture industry, there is a strong desire for an insecticide that does not pose the risk of secondary proliferation caused by spores. By adding and heating
A method of manufacturing by killing the spores has been proposed (Japanese Patent Laid-Open No. 48-22620), but the reason for adopting this manufacturing method is that it naturally complicates the process.

Furthermore, Bacillus thuringiensis has multiple genes encoding insecticidal proteins (Kronstad, J.W. et al.
.

Bacteriol, 154, 419 (1983))
Among the many types of plasmids with different molecular weights, genes encoding insecticidal proteins exist in several plasmids (Aronson, ^, 1.e
tal: Microbio-1 logical Rev
ieim, 50.1 (1986)), and that different insecticidal proteins produced by the same strain have different insecticidal efficacy against the target Lepidoptera larvae (Kondo, S. et al. al:^gric, Bio1
．． Chem. Separates only specific crystal toxins present in a certain ratio from Bacillus thuringiensis culture fluid at a quantitative level.
Since it is impossible to purify and formulate a formulation, the insecticides currently manufactured are composed of a mixture of these, so they are based on crystalline toxins, which are originally the most effective against the target pests. If you think about it, they are in a state of dilution, and in order to achieve a certain effect, the concentration or amount used must be increased.

Composition of the Invention [Means for Solving the Problems] In order to solve the above-mentioned problems, the present inventors conducted intensive studies and developed a mutant strain that lost its spore-forming ability by treatment with a mutating agent such as EMS. Selective production of any insecticidal protein derived from Bacillus thuringiensis variant kurstakii that can solve all of the above problems by creating a mutant strain of B. We succeeded in creating a mutant strain and completed the present invention.

That is, the present invention relates to Bacillus thuringiensis characterized by having lost a part or all of a plasmid carrying a gene encoding an insecticidal protein capable of killing insects of the order Lepidoptera and a spore-forming ability. Cultivation of a B. thuringiensis variant kurstaki mutant strain and a part of a plasmid carrying a gene encoding an insecticidal protein having insecticidal activity against Lepidoptera insects and a Bacillus thuringiensis variant kurstaki mutant strain that has lost spore-forming ability. The present invention relates to an insecticide characterized in that the insecticidal protein obtained by the above method is used as an active ingredient.

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A strain that has lost the ability to form spores can be produced by a known method, that is, by treating bacteria of the genus Bacillus with a mutagen, such as ethyl methanesulfonic acid (EMS), which does not form normal spores. It is possible to create mutants. However, many of the mutants not only do not form spores, but also do not produce crystal toxins at all, or the amount produced is significantly reduced compared to before mutation treatment. However, in order to achieve the desired effect of the present invention, it is necessary not only to not form spores, but also to have the crystal toxin production ability equal to or higher than before the mutation treatment, and the present inventors have long As a result of conducting screening over a period of time, we were able to obtain a mutant strain that lacks spore-forming ability and produces exactly the same amount of crystal toxin after cultivation as before the mutation treatment.

In addition, the present inventors have discovered that the insecticidal properties of several species, including those present on the chromosome, are retained in mutant strains that have lost spore-forming ability and have been created by treatment with mutagens. Among genes encoding proteins (hereinafter referred to as insecticidal protein genes), insecticidal protein genes that are judged to contribute to the production of insecticidal proteins that have a high insecticidal effect against target pests remain in the bacterial body. On the other hand, for those that produce products that have no, low, or unclear insecticidal efficacy against the target pest, the insecticidal protein gene encoding the protein may be partially or partially removed from the bacterial body. By eliminating all of them, a spore-deficient mutant strain of Bacillus thuringiensis was created that selectively produces any insecticidal protein among the insecticidal proteins normally produced by the strain.

The above mutant strain can be produced by a treatment in which the plasmid that is normally carried by the wild-type parent strain is deleted, or a plasmid transfer treatment by conjugation (Mating) between the mutant strain that has lost the plasmid that carries the insecticidal protein gene and the parent strain. It was possible to obtain this by applying

The Bacillus thuringiensis variant kurstaki mutant strain according to the present invention does not form spores and selectively produces insecticidal proteins that are highly effective against target pests, such as the diamondback moth, compared to the parent strain before mutation treatment. Since the intention is to achieve high production, it is a natural consequence that the insecticidal protein gene-carrying plasmid that should be left in the bacterial body will differ depending on the pest that the parties have decided to target. The insecticidal protein gene carrying plasmid is
Any substance that is originally included in the wild type of the mutant strain may be used.

"Effect" According to the present invention, among the insecticidal proteins encoded by different genes in the same strain, the abundance ratio of the protein most effective against the target insect pest per unit amount of crystal toxin is increased. This makes it possible to produce more active insecticides, so that in situations where, for example, crop damage is caused by an outbreak of a particular pest, which is often the case, it is possible to produce a more active insecticide that is highly effective against that pest. It is now possible to supply a formulation that contains a high concentration of crystalline toxin and has a higher specific activity against pests such as the diamondback moth than conventional formulations without increasing production costs. Since it does not contain possible spores, it has an excellent effect in that there is no danger of the spread or proliferation of microorganisms.

"Example" Detailed examples are shown below, but the method according to the present invention is not limited to these examples.

Example 1 0 Mutant Immunization Production Step 1 Suspension of spores of Bacillus thuringiensis var. After cooling, add 0.75 ml of EMS (equivalent to 0.6 M) and shake at 30°C for 2 hours (50 cycles/w).
After aseptic centrifugation (14,000 rpm, 10
minute).

After washing the precipitate several times with sterile physiological saline, add 5 ml of pH 6
, 5 in phosphate buffer. The same suspension was applied to a Petri dish and cultured at 30°C for 1 day. Relatively small and pale white colonies were selected. After 2 days, half of the colonies were transferred to a fresh slant medium (Nutrient Broth-
After culturing at 30°C for 1 day, only those that were observed to grow on the slope were stored at 5°C, and the others were discarded. Meanwhile, remove the remaining half of the colonies on the plate for another 3
Cultured at 0°C for 1 day, suspended in 1 ml of sterile physiological saline, and after heating 0.51111 at 70°C for 20 minutes, Nu
i! in trient-Broth medium! Mi, after solidification, 3
After culturing at 0''C for 2 days, colonies with 0 bacteria, ie, no spore-forming ability, are selected.

Next, the residual liquid from the heat treatment of the suspension of the same colony is examined under a microscope to check for the presence or absence of crystalline toxin production.

As a result of the above test, a spore-deficient strain was obtained which produced crystal toxin to the same extent as before the mutation treatment, and this was used in the experiment described below.

Step 2 The spore-deficient mutant strain obtained above was
ard et al. (J, Bacteriol, 133.8
97 (1978)), a plasmid curing process is performed. i.e. Broth containing 0.002% Sodium Dodecyl Sulfate (SDS) (Log) Ribton, 5g Sodium Chloride, 5g Yeast Extract, 1g Glucose at 12
(dissolved in distilled water) culture with shaking at 42°C for 12 days. After that, the culture solution was diluted appropriately, applied to a plate (L Floss agar medium), and cultured at 30°C for 1 day.
Colonies randomly extracted from the resulting colonies were cultured with shaking at 30°C for 16 hours in test tubes each containing 5 ml of broth medium.

J, Bacteriol, 162.543 (19
85) Perform plasmid extraction according to the method described in ).

The extract is subjected to agarose electrophoresis to analyze the plasmid contained in each colony.

As a result, strain MA31-1 lost a plasmid with a molecular weight of approximately 44Md, and at the same time as 44Md, a plasmid with a molecular weight of approximately 11
An MA31-0 strain was obtained which also lost the 0Md plasmid (
(see Figure 1).

These plasmids contain insecticidal protein genes (Yamamoto, T, et al.
al.

Current Microbiology, 17.
5 (1988)) is retained.

Step 3 Each strain MA31-0 and the original strain before mutation treatment (Bacillus thuringiensis variety Kurstaki HD-1) were
After incubation with shaking at 30° C. for 8 hours in test tubes containing 1 ml of fresh pear broth medium, 1 ml aliquots are transferred to test tubes containing 10 ml of fresh pear broth medium. After standing at 30°C for 4 hours, the appropriately diluted culture solution is applied onto a broth agar plate. After culturing at 30°C for 3 days, bacteria were scraped from colonies in which no sporulation was observed, and the plasmids were analyzed in the same manner as in step 2.

As a result, strain MA312 was obtained which did not contain the l10Md plasmid within its bacterial cells.

Bacillus thuringiensis variant kurstaki mutant strains of the present invention obtained in the above examples M A 31-0, M
A31-1 and MA31-2 are each BTKMA31
-0, BTKMA31 1, and BTKMA31-2, and were sent to the Institute of Microbial Technology, Agency of Industrial Science and Technology, located at 11-3 Higashi, Tsukuba City, Ibaraki, on May 24, 1989, respectively, with the number 10737 of the microbiological research institute. No. 10738, No. 1073
Deposited as No. 9.

The above three strains lack spore-forming ability and are taxonomically the parent strain, except for the qualitative difference in the crystal toxin produced due to the difference in the type of insecticidal protein gene carrying plasmid present in the bacterial body. It is identical to Bacillus thuringiensis var. kurstaki HD-1.

○ Soro M Kenta As a method for producing an insecticide derived from the Bacillus thuringiensis mutant strain MA3m-1,2 obtained according to the present invention, the method for producing an insecticide using the original strain of the present strain before mutation treatment is For example, Dulmag
e, Il, T, :J, Invertebr, Pa
thol, .

22, 273 (1971)).

More specifically, the mutant strain is cultured in a natural medium rich in nitrogen sources, carbon sources, minerals, and vitamins.

Since the production amount of crystal toxin and bacterial cells is greatly affected by aeration and agitation conditions, cultivation should be carried out under sufficient aerobic conditions. The culture temperature is approximately 25-30°C. The number of culture days is:
2 to 4 days is best.

Examples of carbon sources include sucrose, maltose, glucose,
Fructose and molasses are used, and examples of the nitrogen source include corn stew liquor, ammonium sulfate, ammonium chloride, cottonseed flour, yeast extract, soybean flour, and casein melt.

Moreover, minerals, vitamins, and minerals can be replaced with molasses, corn stew liquor, and yeast extract, and if necessary, inorganic salts and vitamins may be further added.

Particularly when performing mass production, deep aeration agitation culture is desirable.

When the crystal toxin-containing portion is separated and collected from the culture solution after completion of the culture, conventional centrifugation methods, filtration methods, etc. can be used. Further, when concentrating and drying the culture solution to form a powder, a conventional concentration method or drying method (eg, spray drying method) may be used.

If the crystalline toxin obtained by the above method contains viable bacteria, it is sterilized using commonly used sterilization techniques, if necessary. For example, physical sterilization methods such as heat treatment, ultrasound treatment, radiation treatment, or chemical sterilization methods such as formalin, hydrogen peroxide, sulfites, chlorine compounds, β-propiolactone, surfactants, ethylene oxide, propylene oxide, etc. Biological sterilization methods include biological sterilization methods, autolysis, phage treatment, and ridzyme treatment, but heat treatment methods and chemical sterilization methods are easier to use industrially.

The obtained crystalline toxin can be formulated into a formulation depending on its purpose of use and dispersion method, and fillers, spreading agents, surfactants, stabilizers, etc. can be added as necessary. It can also be mixed with other agricultural chemicals depending on the purpose.

MA31-1 or MA31- obtained by the above culture method
A bioassay was conducted to examine the insecticidal activity of the 2 culture solution.

In other words, these culture solutions were diluted with distilled water and mixed with artificial feed manufactured by Kyodo Feed Co., Ltd., and fed to 10 3rd instar silkworms or diamondback moth larvae, and the crude protein concentration was determined from the mortality rate after 3 days. Converted to LC. was determined and used as an index of insecticidal activity.

- (The following is a blank space) - Table 1: Insecticidal activity against silkworm When the target insect is silkworm, as shown in Table 1, the insecticidal activity per crude protein in the culture solution using the mutant strain MA31-1 obtained in the present invention The activity exceeded the insecticidal activity per crude protein in the culture solution using the parent strain HD-1.

Table 2: Insecticidal activity against the diamondback moth When the target insect is the diamondback moth, as shown in Table 2, the insecticidal activity in the culture solution using the mutant strain MA31-2 obtained in the present invention is higher than that of the parent strain HD-1. It exceeded the insecticidal activity per crude protein in the culture solution using . Therefore, as seen in these results, by using these mutant strains in the production of insecticides, it is possible to selectively produce high levels of insecticidal proteins that exhibit particularly high insecticidal effects depending on the target pest. became possible.

C.3 Effects of the invention According to the present invention, highly effective crystalline toxins are contained in a high concentration,
Insecticides with higher specific activity than conventional formulations and without germinable spores, i.e., the spread of microorganisms,
It is possible to supply an insecticide that poses no risk of proliferation without increasing production costs, and this has a very large effect on the agrochemical industry and agriculture.

[Brief explanation of the drawing]

Figure 1 shows that plasmid extracts from each strain were subjected to gel electrophoresis, the 0.7% agarose gel was denatured with alkaline, and the plasmids were hypermobilized from the gel to a nitrocellulose membrane by Southern blotting. Kondo, S. et al: Agric. Biol, Chem, 51, 455 (1987).
The insecticidal protein gene was encoded on a nitrocellulose membrane by performing nucleic acid hybridization using a part of the structural gene DNA of the insecticidal toxin of the HD-1 strain shown by the authors as a probe. A plasmid was detected. The number l in the figure is Bacillus thuringiensis kurstakii HD1 strain, and 2 is MA31.
0.3 is an extract from MA31-2 strain. The arrow marked at the right end of the figure indicates the approximate molecular weight of the plasmid encoding the insecticidal protein gene.

Claims (1)

[Scope of Claims] 1. A Bacillus characterized by having lost part or all of a plasmid carrying a gene encoding an insecticidal protein capable of killing insects of the order Lepidoptera, and the ability to form spores.・Thuringiensis var. kurstaki mutant. 2. Obtained by culturing a part of a plasmid carrying a gene encoding an insecticidal protein capable of killing insects of the order Lepidoptera and a Bacillus thuringiensis variant kurstaki mutant strain that has lost the ability to form spores. An insecticide characterized by containing insecticidal protein as an active ingredient.