JP2842578B2 - New microorganisms and pesticides - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel microorganism belonging to the genus Bacillus thuringiensis cellova japonensis and an insecticide derived from the novel microorganism.

[0002]

2. Description of the Related Art Conventionally, Bacillus thuringiensis
Among strains known from the genus Serovar yaponensis, those producing insecticidal proteins against larvae of lepidopteran insects are known. Also known are Bacillus thuringiensis bacteria, such as Bacillus thuringiensis San Diego and Bacillus thuringiensis tenebrionis, which kill a member of the beetle, a member of the Colorado potato beetle and a beetle beetle, the white beetle beetle, J. Appl. Ent. 104 (1987), 417-424).

[0003]

However, since no strain of the genus Yaponensis produces toxin proteins other than insecticidal proteins against larvae of lepidopteran insects, insecticides against insects other than lepidoptera are not known. The agent was not available. Bacillus thuringiensis san diego, Bacillus thuringiensis tenebrionis and the like do not have an insecticidal effect on the larvae of the larvae of P. persica, which are large pests such as grass, taro, sweet potato, and peanut. The present inventors, a new strain belonging to the genus Bacillus thuringiensis cellova japonensis,
An object of the present invention is to produce an insecticidal protein against Coleoptera larvae other than lepidopteran insects, and to provide a novel microorganism and a novel microorganism-derived insecticide that can be effectively used.

[0004]

Means for Solving the Problems The novel microorganism of the present invention comprises:
Bacillus thuringiensis belonging to Bacillus thuringiensis cellova japonensis and having the ability to produce insecticidal toxin proteins against Coleoptera larvae
It has the features of Selova Yaponensis strain buoy. And, Bacillus thuringiensis cellova, which is a strain of a novel microorganism belonging to the genus Bacillus thuringiensis cellova japonensis of the present invention.
Yaponensis strain buoy (Bacillus thuri)
ngiensis serovar japonensis strain Buibui) has been deposited at the Research Institute of Microbial Industry and Technology of the National Institute of Advanced Industrial Science and Technology as Microtechnical Laboratory No. 3465 (FERM BP-3465).
In addition, the novel microorganism-derived insecticide of the present invention, Bacillus
It contains as an active ingredient a toxin protein produced by Thuringiensis cellova japonensis strain buoy.

[0005]

(Bacteriological properties)

1. Growth state in each medium The present bacterium can grow in almost all mediums that can be used for cultivation of normal bacteria, and can produce toxin protein to some extent. As shown in FIGS. 1 and 2, normal growth was shown in a typical medium such as NYS, L-broth and broth medium. That is, the cell number began to increase logarithmically in several hours and stopped increasing in 24 hours. Toxins appear with a slight delay in increasing cell numbers. The toxin amount is 200 to 3000 μg / m when the main band is measured at 130 kDa.
1 medium. By searching for a suitable medium, Bt
Production rates of 5000 μg / ml, which have been reached in fungi, are also possible. 2. Morphological Properties As shown in FIG. 3 and FIG. 4, the resulting colonies have the characteristics of ordinary Bacillus bacteria having a glossy surface on an agar medium, not bulging, but spreading thinly on the agar surface and having a rough surface. The color of the colonies is light beige. When observed using a scanning microscope, as shown in FIGS. 5 and 6, Bacillus thuringiensis cellova yaponensis (hereinafter abbreviated as “yaponensis strain”) and Bacillus thuringiensis cellobar yaponensis strain buibu (hereinafter “buoy buoy strain”) Abbreviated as), spherical crystal proteins were observed. These are different from bipyramid-type crystals commonly found in other Bt bacteria that exhibit insecticidal activity against lepidopteran larvae. 3. Biochemical properties The following tests were performed while comparing the biochemical properties of the present buoy buoy strain with those of the conventional Yaponensis strain. (Test Example 1) Serotyping using an antibody derived from a flagellar antigen Using an antibody against the flagellar protein of Bacillus sp.
This is a method of identification using a flagellar protein of an unknown bacterium as an antigen using an antigen-antibody reaction. The Yaponensis strain is a serotyped H23 type subtype that has already been certified (J. Invertebr. Pathol. 32, 303-309, 197).
8; J. Invertebr. Pathol. 48129-130, 1986). In contrast, the buoy buoy strain reacts with the H antigen of the Yaponensis strain.
And these properties are serologically equivalent to the Yaponensis strain. Therefore, taxonomically, it belongs to the same subspecies as the Yaponensis strain. The details of the experiment are shown below. (1) Preparation of Flagellar H Serum Forty known H antigen reference strains of Bacillus thuringiensis were used. Bacteria with good motility were selected using Craigie tubes (0.5% semi-fluid agar medium), and formalin-killed bacteria were prepared using the bacterium and immunized rabbits. H
Serum was prepared by absorbing antibodies against the corresponding Bacillus thuringiensis cell antigen from each antiserum. The cell antigen was heated at 100 ° C. to remove the flagella and adjusted. (2) Identification of H antigen The serotype of the H antigen was determined by placing a glass agglutination reaction (Ohba and Ai
zawa, I. Invertebr. Pathol., 32, 303-309, 1978). The agglutination titer of H serum was determined by a test tube agglutination reaction (Ohba and Aizawa, 1978). (3) Results The yaponensis strain was specifically agglutinated only by the serum against the reference strain of Serovarjaponensis (H antigen 23) among the standard sera containing only the known 40 types of flagellar antibodies. The H serum against the buoy buoy strain specifically aggregated only the yaponensis strain. The agglutination titer for the corresponding homoantigen of the Yaponensis H serum was 12,800-fold and the agglutination titer for the buoy strain was 6,400-fold. The agglutination titer for homozygous Vbuoy strain H serum was 12,800.
The agglutination titer for the Yaponensis reference strain is:
It was 6,400 times. Therefore, both strains are judged to be the same. Test Example 2 Insecticidal Spectrum of Crystal Proteins Produced by Yaponensis and Vegetative Strains As shown in Table 1, the insecticidal proteins produced by the buoy vegetative strain are the insects of the order Coleoptera, Anomala cuprea H.
ope) at a concentration of 0.125 to 12.5 µg / ml, but the insecticidal protein produced by the Yaponensis strain did not show insecticidal activity even at a concentration of 100 µg / ml. As shown in Table 2, among the lepidopteran insects, those of the Yaponensis strain are Japanese moth (Plutella xylostella),
The larvae of Adoxophyes sp. And Bombyx mori showed high activity, while the buoy strain exhibited very low or little activity.
These results suggest that both crystalline proteins have different compositions. Therefore, it can be seen that both strains cannot be said to be identical strains.

[0006]

[Table 1]

[0007]

[Table 2]

Test Example 3 Electrophoresis of Insecticidal Proteins Accumulating in Cells of Yaponensis and V. buoy strains The target microorganism produces spherical crystalline proteins in the cells similarly to the Yaponensis strain (FIG. 5, FIG. 5). 6). This crystalline protein is prepared by a conventional method (Goodman, NS, RJ Gottfried a
nd MHRogoff, J. Bacteriol. 94: 485, 1967), isolated from the culture medium, purified, dissolved in a 0.4 N alkaline solution, and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE). FIG.
As shown in the figure, the main band of the Yaponensis strain was about 76 kilodalton (kDa), and another 52 kDa was observed.
On the other hand, the main band of the buoy buoy strain is 130 kDa and the other band is 52 kDa.
Da, 45 kDa was observed. These electrophoretic patterns clearly show that the components constituting both crystal proteins are different. (Test Example 4) Difference in assimilation in culture of Yaponensis strain and V. buoy strain As shown in Table 3, the Yaponensis strain could assimilate glucose, salicin, maltose, could not assimilate mannose, and cellobiose could not be assimilated. Some assimilation. The buoy strain was able to utilize all of the above compounds. The above-mentioned points indicate that the buoy buoy strain is taxonomically classified as a Yaponensis strain by serotype, but is distinctly different from the Yaponensis strain.

[0009]

[Table 3]

(Test Example 5) Insecticidal activity of the buoy strain against the beetle insect Douganebuui As described above, the buoy buoy strain exhibited a very high insecticidal activity, which has not been reported yet, by using A. cupii (A. cup).
rea Hope). The insecticidal activity of the buoy buoy strain was investigated using the larvae of the first to third larvae. The activity was evaluated as follows. 2 ml of water containing an insecticidal component was added to 2 g of the dried humus, and the mixture was put in a plastic cup, and larvae were placed one by one and bred for a predetermined time. As the insecticidal component, a culture broth (i.e., a liquid containing microbial cells) in which the buoy strain was cultured, and a product obtained by isolating and purifying a crystalline toxin protein from the culture broth were used to examine the insecticidal activity of each. The mortality is a value obtained by dividing the number of dead larvae by the total number of larvae. According to this, as shown in FIG. 8, it was shown how the mortality rate changes with time depending on the amount of the insecticidal component (toxin) composed of the culture solution.
Even with a low toxin dose of 25 μg / ml, 12.5
It has been found that a high toxin dose of μg / ml can achieve 100% mortality, but at low concentrations, all test insects take twice as long to die. In addition, con in the figure
The trol indicates a change when only water containing no toxin is given. As for the insecticidal component comprising the isolated and purified crystalline protein, as shown in Table 4, the crystalline protein alone exhibited an insecticidal activity. However, the insecticidal activity could not be detected at 0.1 μg / ml of the crystal,
When the culture containing approximately 0.1 μg / ml of the 130 kDa protein was fed to the dolphin buoy, as described above (FIG. 8), the mortality was slow but 100%. This is not due to the loss of activity due to denaturation of the protein during the process of purifying the crystalline protein, but not to the fact that the spores present in the cells show a higher activity in the sclerophyllis in cooperation with the crystalline protein. Seems to be. Therefore, the insecticide preparation may contain cells.

[0011]

[Table 4]

(Test Example 6) Insecticidal effect on larvae of other Coleoptera insects, as shown in Table 5, the buoy buoy strains other than Douganebuui buddha (Anomala rufocuprea Motschulsky),
It also showed high insecticidal activity against Aedala schoenfeldti Ohaus. Therefore, it can be expected to show an insecticidal effect on some other scarab beetle larvae. Therefore, the target of the insecticide is not limited to these three kinds of Coleoptera larvae.

[0013]

[Table 5]

The first larva was used except for the third larva of Himekogane. The crystals are purified from cells cultured in NYS, and the number of test animals is 10. The above control shows the result (comparative experiment) when only water was given without toxin.

Test Example 7 Insecticidal Effect Against Other Coleoptera Insects First-instar larva of Coleoptera, Anomala albopilosa, first-instar larva of Anomala daimiana, first-instar larva of beetle (Minela splendens), (Popillia japonica) and the second instar larva of Blitopertha orientalis were used to investigate the insecticidal activity of the buoy strain against each larva. For each test insect, an adult was collected from the field, and young larvae of larvae generated from eggs bred temporarily and laid in commercial mulch were used.
The test method is as follows: 1 g of humus which has been sterilized by dry heat in a dry oven at 160 ° C. for 60 minutes is weighed into a plastic cup with a lid having a capacity of about 30 ml. Multiple larvae containing one larva were prepared, and these were bred in a constant temperature room at 25 ° C.
A method was employed in which the mortality rate after 4 and 21 days was investigated and the buoy buoy titer was tested. 〔Test results〕

[0016]

[Table 6]

The numbers of the test insects of Sakurakogane and Semaedaragane are 8 and 5, respectively, and all other insects are 10 in number.

[Conclusion] As shown above, the buoy buoy strain exhibited insecticidal activity against Aedogane, Sakurakogane, Scarabaeidae, Mamekogane and Semaedaragane. The mortality after 21 days in the case of Sakurakogane is still as low as 70% as compared to other insect larvae, but it is clear that no weight gain is observed and the mortality will follow. Therefore, although a slight delay in the effect is observed, it can be determined that the effects of stopping eating and death are completely equivalent. Especially Japanese bee
The pesticidal properties of the beetle, which is a major problem in the United States, called tle, are particularly important. Although the activity against several Coleoptera was identified, the target was found not only in the genus Anomala but also in the genus Popillia, Minela, and Blitopertha. This indicates that the target pests of this fungus were the species shown in Tables 5 and 6. Not limited to only Anomala, Popillia, Minela, Bl
It suggests that the genus ithopertha extends to Coleoptera insects other than the above, or broadly other genera. (Test Example 8) Activity of beta-exotocin Some bacteria belonging to the genus Bacillus secrete beta-exotoxin which is a nucleotide derivative in the medium. It has an insecticidal effect like the toxin protein. Beta-exotoxin has a teratogenic effect on housefly larvae, and is used to evaluate beta-exotoxin activity. However, as shown in Table 7, even if the culture supernatant was prepared from the culture medium of the buoy species and fed to the house fly according to a conventional method, neither the pupation rate nor the emergence rate was affected, and the teratogenicity of the buoy strain was not shown. . When the treated medium of the above-mentioned buoy buoy was fed to Dougane buoy, the larva did not die even after 14 days as shown in Table 8. The results of this experiment indicate that the insecticidal effect of the buoy buoy strain on D. serrata was not derived from beta-exotoxin. In other words, it can be seen that beta-exotoxin does not exist so as to affect the above-mentioned experimental results.

[0019]

[Table 7]

[0020]

[Table 8]

(Note that the buoy medium described above refers to the remaining medium obtained by removing the strain from the medium by centrifugation.)

[0022]

The effectiveness of the microorganism of the present invention is determined by culturing the microorganism to produce a crystalline protein exhibiting an insecticidal activity against larvae of Coleoptera, which is not found in conventional Yaponensis strains. By formulating an insecticide that is selectively active against Coleoptera larvae, derived from a novel microorganism containing the toxin protein as an active ingredient, it is safer and cheaper than chemically produced insecticides. Become.

[0023]

EXAMPLES The buoy strain can easily grow in a medium commonly used for culturing bacteria, such as L-broth and nutrient broth, and produce spores and crystal proteins. In producing insecticidal ingredients including
A medium with high productivity was studied. First, 3.3 × 10 ⁵ spores were inoculated on a 9 cm Petri dish agar medium, and
After a day, the crystalline protein produced was observed under a microscope.
As a result, MnSO ₄ (10 ⁻³⁾ was added to L-broth as follows.
The medium to which M) was added produced the best, and the following order was used. L-broth + MnSO ₄ > spitizen + amino acid> L
-Broth>PGSM> spitizen + casamino acid + vitamin> spitizen + casamino acid>NYS> NYS + casamino acid Each medium has the following composition. L-broth: Each component content is 10 g of tryptose, 5 g of yeast extract and 5 g of salt per liter, pH = 7.
18 to 7.2. Spichizen: Each component content is 1 phosphoric acid per liter
14 g of potassium hydrogen, 6 g of potassium dihydrogen phosphate, 2 g of ammonium sulfate
g, magnesium sulfate 7H ₂ O 0.2 g, sodium citrate 1 g, glucose 5 g, pH = 7.0. NYS: 1.25 g of nutrient broth, 1.25 g of tryptone, 0.5 g of yeast extract, 0.206 g of calcium chloride 2H ₂ O , salt per liter
0.407 g of magnesium chloride 6H ₂ O, manganese chloride 4
H ₂ O0.02g, iron sulfate 7H ₂ O0.0004g, sulfuric acid
Zinc 7H ₂ O 0.0004 g ammonium sulfate 0.00
04 g , pH = 7.2. NYS + casamino acid: a medium obtained by adding 2.0 g of casamino acid to the above-mentioned NYS medium. pH = 7.2 Next, when formulating an insecticide for larvae of Coleoptera insects using the insecticidal crystal protein produced by the present bacterium, the present microorganism is prepared by using the above-mentioned various media or fishmeal, soybean meal, etc. Culture using a solid medium, corn syrup, starch industry such as constere, waste from the sugar-making process, etc., and concentrate the cells cultivated by such various methods into a creamy product. This is appropriately diluted in water or the like to make an insecticide for spraying. The creamy product may be mixed with a preservative, a bulking agent, and the like according to a conventional method, or may be made into a powdery state by using a spray dryer thereafter. The above-described method is performed using the cells producing the toxin protein itself, but it is also possible to use the crystalline protein by culturing until the cells undergo autolysis. The concentration of the crystal can be performed by centrifugation or using a membrane. The preparation thus prepared is used as a viable preparation for the bacterium to produce spores. The toxin protein produced by this bacterium does not show toxicity to silkworms, and therefore is a viable bacterial preparation that does not have any impact on sericulture even when used as a viable bacterial preparation having spores. In addition, the spores can be killed by using an appropriate compound and used as a killed bacterial preparation. Explaining the method of spraying the formulation, the larva of the target Coleoptera insects usually inhabit the soil, therefore, the insecticide containing the fungus as an active ingredient, sprayed in the soil, or You can plow it with a cultivator immediately after spraying with mulch, etc.
You can plow it directly by spraying. Further, using an automatic or manual syringe-like device, the suspension of the insecticide can be directly injected into the soil. For this purpose, a fully automatic injector may be installed in a tiller or the like. . Incidentally, the toxin protein produced by the buoy strain can also be produced by genetic engineering using a gene encoding the protein.

[Brief description of the drawings]

FIG. 1 is a graph showing a growth curve of a buoy strain.

FIG. 2 is a graph showing a growth curve of a buoy strain.

FIG. 3 is a photograph showing a form of a living thing.

FIG. 4 is a photograph showing the form of an organism

FIG. 5 is a scanning electron micrograph showing the morphology of an organism .

FIG. 6 is a scanning electron micrograph showing the morphology of an organism .

FIG. 7 is a photograph showing electrophoresis .

FIG. 8 is a graph showing a time-course mortality curve of Douganebuui larvae.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI (C12N 1/20 C12R 1:07) (C12P 21/00 C12R 1:07) (72) Inventor Nobukazu Suzuki Koyodai, Ryugasaki-shi, Ibaraki 5-6 Tsukuba Laboratory, Kubota Technology Development Laboratory Co., Ltd. (72) Inventor Katsutoshi Ogiwara 5-6 Koyodai, Ryugasaki-shi, Ibaraki Prefecture Tsukuba Laboratory, Technology Research Laboratory Kubota Co., Ltd. (72) Kazuhiro Sanaka, Inventor Ibaraki (6) Inventor Hidetaka Hori 5-6 Koyodai, Ryugasaki-shi, Ibaraki Pref. Tsukuba Laboratory (72) Inventor Asano Shoji 5-7 Koyodai, Ryugasaki, Ibaraki Pref. Tsukuba Laboratory, Kubota Technology Development Laboratory Co., Ltd. (72) Inventor Kawa Tadaaki Ibaraki Prefecture Ryugasaki City Koyodai 5-6 Kubota Corporation Technology Development Institute Tsukuba Research chamber (58) investigated the field (Int.Cl. ^6, DB name) C12N 1/00 - 7/08 BIOSIS (DIALOG ) WPI ( DIALOG)

Claims (2)

(57) [Claims] 1. A bacterium belonging to Bacillus thuringiensis serovar japonensis and having an ability to produce an insecticidal toxin protein against larvae of Coleoptera.
Thuringian cis cellova japonensis strain buoy (Microtechnical Engineering Co., Ltd. No. 3465). 2. An insecticide containing as an active ingredient a toxin protein produced by Bacillus thuringiensis cellova japonensis strain buoy.