JPH07179A - Novel microorganism and insecticide - Google Patents

Abstract

PURPOSE:To obtain an insecticide containing a novel crystalline toxin protein which is effective against Coleoptera larvae with higher safety and inexpensiveness than those of chemically produced insecticides. CONSTITUTION:Bacillus thuringiensis serovar japonensis strain Buibui producing insecticidal protein against Coleoptera larvae, and the insecticide contains, as an active ingredient, a toxin protein produced by Bacillus thuringiensis serovar japonensis strain Buibui.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel microorganism belonging to the genus Bacillus thuringiensis serovar japonensis and an insecticide derived from the novel microorganism.

[0002]

2. Description of the Related Art Conventionally, Bacillus thuringiensis
Strains known to the genus Serovar japonensis are known to produce an insecticidal protein for larvae of Lepidoptera insects. In addition, Bacillus thuringiensis bacteria such as Bacillus thuringiensis San Diego and Bacillus thuringiensis tenebrionis, which kill the beetle companion, Colorado potato beetle and the beetle companion, the beetle Tadpole beetle, are known (J. Appl. . Ent. 104 (1987), 417-424).

[0003]

However, since no strains of the genus Japonensis that produce toxin proteins other than insecticidal proteins for larvae of Lepidoptera insects are known to be insecticidal to insects other than Lepidoptera. It was not available as a drug. In addition, Bacillus thuringiensis san diego, Bacillus thuringiensis tenebrionis and the like have no insecticidal effect on the larvae of Dougane buoy, which is a major pest of turfgrass, taro, sweet potato, peanut and the like. The present inventors have confirmed that a new strain belonging to the genus Bacillus thuringiensis serovar japonensis is
The present invention finds that an insecticidal protein is produced against beetle larvae other than Lepidoptera insects, and aims to provide a novel microorganism and an insecticide derived from the novel microorganism that can be effectively used.

[0004]

[Means for Solving the Problems] The novel microorganism of the present invention is
Bacillus thuringiensis, which belongs to Bacillus thuringiensis serovar japonensis and has the ability to produce an insecticidal toxin protein against Coleoptera larvae
It is characterized by Serobar, Japonensis, Strain, and Buoy. And Bacillus thuringiensis serovar, which is one of the novel microorganisms belonging to the genus Bacillus thuringiensis serovar japonensis of the present invention.
Japonensis strain buoy (Bacillus thuri
ngiensis serovar japonensis strain Buibui) has been deposited with the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology as the Micro Engineering Research Article No. 3465 (FERM BP-3465).
Further, the insecticide derived from the novel microorganism of the present invention is Bacillus
It contains a toxin protein produced by Thuringiensis serovar japonensis strain buoy as an active ingredient.

[0005]

[Mycological properties]

1. Growth state in each medium The bacterium can grow in almost all the mediums that can be used for culturing ordinary bacteria, and can produce toxin protein to some extent. As shown in FIGS. 1 and 2, normal growth was shown in a representative medium of NYS, L-broth and broth medium. That is, the number of cells started to increase logarithmically within several hours and stopped increasing within 24 hours. Toxin appears a little later than the increase in cell number. The amount of toxin is 200 to 3000 μg / m when the main band 130 kDa is measured.
1 medium. By searching for the appropriate medium, Bt
The production amount of 5000 μg / ml that has been reached in bacteria is also possible. 2. Morphological Properties As shown in FIGS. 3 and 4, the resulting colonies have the characteristics of a normal Bacillus bacterium, which has a glossy surface on the agar medium, does not rise, spreads thinly on the agar surface, and has a rough surface. The color of the colony is light beige. When observed with a scanning microscope, as shown in FIGS. 5 and 6, Bacillus thuringiensis serovar japonensis (hereinafter abbreviated as Japonensis strain) and Bacillus thuringiensis serovar yaponensis strain buoy (hereinafter buoy strain) (Abbreviated as)), spherical crystal proteins were observed. These are different from the bipyramidal crystals that are often found in other Bt bacteria that show insecticidal activity against Lepidoptera larvae. 3. Biochemical Properties The following tests were carried out while comparing the biochemical properties of the buoy buoy strain, which is the bacterium, with that of the conventional Japonensis strain. (Test Example 1) Serotyping Using Antibodies Derived from Flagella Antigen Using antibodies to flagella proteins of Bacillus,
This is a method of identifying by using an antigen-antibody reaction using a flagellar protein of an unknown bacterium as an antigen. The japonensis strain is a subspecies that has already been certified as H23 type by serotyping (J. Invertebr. Pathol. 32,303-309, 197).
8; J. Invertebr. Pathol. 48129-130, 1986). On the other hand, the buoy buoy strain reacts with the H antigen of the Japonensis strain.
And these properties are serologically equivalent to the Japonensis strain. Therefore, taxonomically, it belongs to the same subspecies as the Japonensis strain. The details of the experiment are shown below. (1) Preparation of flagella H serum 40 known H antigen strains of Bacillus thuringiensis were used. Bacteria with good motility were selected using a Craigie tube (0.5% semi-solid agar medium), and killed formalin bacteria were prepared using the bacteria to immunize rabbits. H
Serum was prepared by absorbing an antibody against the corresponding Bacillus thuringiensis cell antigen from each antiserum. The bacterial cell antigen was prepared by heating at 100 ° C. to remove the flagella. (2) Identification of H-antigen The serotype of H-antigen is based on the glass-aggregation reaction (Ohba and, Ai
Sawa, I. Invertebr. Pathol., 32, 303-309, 1978). The agglutination titer of H serum was quantified by a test tube agglutination reaction (Ohba and Aizawa, 1978). (3) Results The Japonensis strain was specifically aggregated only by the serum against the reference strain of Serovar japonensis (H antigen 23) among the standard sera containing only 40 known flagella antibodies. Further, the H serum against the buoy buoy strain specifically aggregated only the Japonensis strain. The agglutination titer of the Japonensis H serum to the corresponding homologous antigen was 12,800 times and that of the buoy buoy strain was 6,400 times. The agglutinin titer of homologue of buoy buoy strain H serum is 12,800.
And the agglutination index for the Japonensis reference strain is
It was 6,400 times. Therefore, both strains are considered to be of the same class. (Test Example 2) Insecticidal spectrum of crystal proteins produced by Japonensis strains and buoy buoy strains As shown in Table 1, the insecticidal protein produced by buoy buoy strains is the Coleoptera insects Anomala cuprea HV.
ope) at a concentration of 0.125 to 12.5 μg / ml, the insecticidal protein produced by the Japonensis strain did not show insecticidal activity even at a concentration of 100 μg / ml. As shown in Table 2, Japonensis strains include the diamondback moth (Plutella xylostella) among Lepidoptera insects,
High activity was observed in larvae of Adoxophyes sp. And silkworm (Bombyx mori), while buoy buoy strain showed very low activity 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 exactly the same strain.

[0006]

[Table 1]

[0007]

[Table 2]

(Test Example 3) Electrophoresis of Insecticidal Protein Accumulating in Cells of Japonensis Strains and Buibui Strains The title microorganism produces intracellular spherical crystalline proteins as in the Japonensis strains (FIG. 5, FIG. 5). 6). This crystalline protein was 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 0.4N alkaline solution, and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE). Figure 7
As shown in Fig. 5, the main band of the japonensis strain was about 76 kilodaltons (kDa), and 52 kDa was observed elsewhere.
On the other hand, the main band of buoy buoy is 130kDa and the other 52k
Da and 45 kDa were recognized. These electrophoretic patterns clearly show that the constituents of both crystal proteins are different. (Test Example 4) Difference in assimilability in culture of Japonensis strain and buoy buoy strain As shown in Table 3, the Yaponensis strain assimilates glucose, salicin, and maltose, but cannot assimilate mannose, and cellobiose Somehow assimilated. The buoy 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 japonensis strain by the serotype, but is clearly different from the japonensis strain.

[0009]

[Table 3]

(Test Example 5) Insecticidal activity of buoy buoy strain against Coleoptera insect Dougane buoy buoy As described above, the buoy buoy strain has a very high insecticidal activity which has not been reported yet.
rea Hope). The insecticidal activity of the buoy buoy strains was investigated using larvae of the first to third instar Douga Nebu buoy. The activity was evaluated as follows. 2 ml of water containing an insecticidal component was added to 2 g of dried leaf soil, put in a plastic cup, and larvae were placed one by one and bred for a predetermined time. As the insecticidal component, a culture solution in which a buoy buoy strain was cultured (that is, a solution containing bacterial cells) and a crystalline toxin protein isolated and purified from the culture solution were used to examine the insecticidal activity of each. The mortality rate is a value obtained by dividing the number of dead larvae by the total number of larvae. As shown in FIG. 8, it was shown that 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 was found that 100% mortality was obtained even with a high toxin dose of μg / ml, but at low concentrations it took twice as long for all test worms to die. In addition, con in the figure
"trol" indicates a change when only water containing no toxin is given. Regarding the insecticidal component consisting of the isolated and purified crystalline protein, the crystalline protein alone showed insecticidal activity as shown in Table 4. However, no insecticidal activity could be detected with 0.1 μg / ml of crystals,
When the above culture containing approximately 0.1 μg / ml of the 130 kDa protein was fed to Douganebu buoy, it showed a slow but 100% mortality rate as already described (FIG. 8). This is not because the activity is lost due to denaturation of the protein in the process of purifying the crystalline protein, but the spores present in the bacterial cells do not show higher activity in cooperation with the crystalline protein in Dougane buoy. It seems that Therefore, the insecticidal preparation may contain cells.

[0011]

[Table 4]

(Test Example 6) Insecticidal effect of buoy buoy strain on larvae of other beetle insects As shown in Table 5, the buoy buoy strain is not only Dougane buoy buoy, but also Anemala rufocuprea Motschulsky,
It also showed a high insecticidal activity against the white-spotted beetle (Anomala schoenfeldti Ohaus). Therefore, it can be expected to show an insecticidal effect against some other beetle larvae. Therefore, the target of the insecticide is not limited to these three types of Coleoptera larvae.

[0013]

[Table 5]

The 1st instar was used except for the 3rd instar larva of the Japanese beetle. The crystals were purified from the cells cultured in NYS, and the number of test animals was 10. The above-mentioned control shows the result (comparative experiment) when only water was given without giving toxin.

(Test Example 7) Insecticidal effect against other Coleoptera insects First-instar larvae of Coleoptera, Anomala albopilosa, first-instar larvae of Anomala daimiana, first-instar larvae of Mina splendens, beetle beetle The insecticidal activity of the buoy buoy strain against each larva was investigated using the 1st instar larvae of (Popillia japonica) and the 2nd instar larvae of Blattopertha orientalis. For each test insect, an adult one was collected from the field, temporarily bred in a commercially available leaf soil, and used as a young larva of the larvae generated from the eggs laid.
The test method was as follows: Weigh 1 g of leaf-leaf sterilized by dry heat sterilization in a dry oven at 160 ° C for 60 minutes into a plastic cup with a lid of about 30 ml in volume, mix with a buoy buoy culture of a predetermined concentration, and stir it thoroughly. Prepare multiple larvae containing one and cultivate them in a constant temperature room at 25 ° C.
The mortality rate after 4 and 21 days was investigated to test the buoy buoy titer. 〔Test results〕

[0016]

[Table 6]

[0017] The number of test insects of Prunus chinensis and Scorpionidae is 8 and 5, respectively, but the others are 10 in all.

[Conclusion] As shown above, the buoy buoy strain exhibited insecticidal activity against the blue-spotted beetle, the cherry beetle, the scarab beetle, the beetle, and the beetle. The mortality rate after 21 days in the case of Bombyx mori is as low as 70% as compared with other insect larvae, but no increase in body weight is observed at all, and it is clear that the mortality will occur thereafter. Therefore, although a slight delay in the effect is observed, the effects of stopping feeding and dying can be judged to be exactly the same. Especially Japanese bee
The insecticidal properties against the Japanese beetle, tle, which has become a major problem in the United States called tle, is particularly important. Although the activity against some Coleoptera was identified above, the target was found in the genera Popillia, Minela, and Blitterpertha in addition to the genus Anomala. The target pests of this fungus are the species shown in Table 5 and Table 6. Not limited to only Anomala, Popillia, Minela, Bl
It is suggested that ithopertha extends to other species than the above, or broadly, other Coleoptera insects of other genera. (Test Example 8) Activity of beta-exotocin Among bacteria belonging to the genus Bacillus, some strains secrete beta-exotoxin, which is a nucleotide derivative, into the medium. It has an insecticidal effect similar to the toxin protein. Beta-exotoxin exerts a teratogenic effect on the housefly larva, and the beta-exotoxin activity is evaluated based on it. However, as shown in Table 7, even if the culture supernatant was prepared from the medium of the buoy buoy strain according to a conventional method and given to the house fly, neither the pupation rate nor the emergence rate was affected, and the territory of the buoy buoy strain was not shown. . When the treatment medium of the buoy buoy strain was fed to Dougane buoy buoy, the larvae did not die after 14 days as shown in Table 8. The results of this experiment indicate that the insecticidal effect of the buoy buoy strain on the Spodoptera littoralis is not derived from beta exotoxin. In other words, it can be seen that beta-exotoxin does not exist enough to affect the above-mentioned experimental results.

[0019]

[Table 7]

[0020]

[Table 8]

(The above buoy medium means the remaining medium obtained by removing the strain from the medium by centrifugation.)

[0022]

The effectiveness of the microorganism of the present invention is as follows: by culturing the same, a crystalline protein having insecticidal activity against larvae of Coleoptera insects, which is not present in conventional Japonensis strains, is produced, and the produced crystalline It is derived from a novel microorganism that contains the toxin protein as an active ingredient, and by preparing an insecticide that is selectively active against Coleoptera larvae, it is safer and cheaper than chemically produced insecticides. Become.

[0023]

[Examples] The buoy buoy strain is easily grown in a medium such as L-broth or nutrient broth which is commonly used for culturing bacteria to produce spores and crystal proteins. In producing insecticidal ingredients including
The medium with high productivity was examined. First, 3.3 × 10 ⁵ spores were inoculated on an agar medium of 9 cm Petri dish to obtain 10
The crystalline protein produced after the day was observed microscopically.
As a result, MnSO ₄ (10 ⁻³⁾ was added to L-broth as follows.
The medium to which M) was added had the best production, and the following order was as follows. L-broth + MnSO ₄ > spicyzen + amino acid> L
-Broth>PGSM> spicyzen + casamino acid + vitamin> spicyzen + casamino acid>NYS> NYS + casamino acid Each medium has the following composition. L-broth: The content of each component was 10 g of tryptose, 5 g of yeast extract, 5 g of salt per liter, and pH = 7.
18 to 7.2. Spicysen: Each component content is 1 liter of phosphoric acid per liter.
14 g of potassium hydrogen, 6 g of potassium dihydrogen phosphate, ammonium sulfate 2
g, magnesium sulfate 7H ₂ O 0.2 g, sodium citrate 1 g, glucose 5 g, and pH = 7.0. NYS: Content of each component per liter is 1.25 g of nutrient broth, 1.25 g of tryptone, 0.5 g of yeast extract, 10.3 g of calcium chloride 2H ₂ O, 60.3 g of magnesium chloride 6H ₂ O, 4H _{2 of} manganese chloride.
O1.0g, iron sulfate 7H ₂ O 0.02g, zinc sulfate 7H ₂
At 0.02 g O, pH = 7.2. NYS + casamino acid: Casamino acid 2.
Medium with 0 g added. pH = 7.2 Next, in formulating an insecticide for the larvae of Coleoptera insects using the insecticidal crystal protein produced by the present bacterium, the present microorganism is used in various media described above, fish meal, soybean meal, etc. Culture using solid medium, corn syrup, starch industry such as Consteep, waste in the sugar making process, etc., and concentrate the bacterial cells cultivated by such various methods into a creamy product. , This is used as an insecticide for appropriate dilution by spraying with water or the like. The creamy product may be mixed with a preservative, a bulking agent and the like according to a conventional method, or may be powdered by using a spray dryer after this. The above method is carried out using the cells producing the toxin protein itself, but it is also possible to use only the crystalline protein by culturing until the cells undergo autolysis. Concentration of the crystal can be performed by centrifugation or using a membrane. The thus-prepared preparation is used as a live bacterial preparation because the bacterium produces spores. The toxin protein produced by this bacterium does not show toxicity to silkworm, and therefore, it is a viable bacterial preparation which has no impact on sericulture even when used as a viable bacterial preparation having spores. It is also possible to use a suitable compound to kill spores and use it as a killed cell preparation. Explaining the method of spraying the above formulation, the larvae of the target Coleoptera insect usually inhabit the soil, and therefore, the insecticide containing the fungus as an active ingredient is sprayed in the soil, or Immediately after spraying with mulch, you can plow with a tiller,
You can also spray it directly and plow it in. Further, the suspension of the above insecticide can be directly injected into the soil by using an automatic or manual injector-like device, and therefore a fully automatic injector may be installed in a tiller or the like. . The toxin protein produced by the buoy buoy strain can also be produced by genetic engineering using a gene encoding the protein.

[Brief description of 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 colonies of buoy buoy strain in LB medium.

FIG. 4 is a photograph showing colonies of a buoy strain in each medium.

FIG. 5: Scanning electron micrograph of Japonensis strain

FIG. 6 Scanning electron micrograph of buoy strain

FIG. 7 is a photograph showing sodium dodecyl sulfate polyacrylic amide gel electrophoresis.

FIG. 8 is a graph showing a time-course death curve of Larvae of Rhododendron

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[Procedure amendment]

[Submission date] February 5, 1992

[Procedure Amendment 1]

[Document name to be corrected] Drawing

[Correction target item name] All drawings

[Correction method] Change

[Correction content]

[Figure 1]

[Fig. 2]

[Figure 3]

[Figure 4]

[Figure 5]

[Figure 7]

[Figure 6]

[Figure 8] ─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] October 29, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] Brief description of the drawing

[Correction method] Change

[Correction content]

[Brief description of 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 the morphology of living things

FIG. 4 is a photograph showing the morphology of organisms

FIG. 5: Scanning electron micrograph showing the morphology of living things

FIG. 6 Scanning electron micrograph showing the morphology of living things

FIG. 7 is a photograph showing electrophoresis .

FIG. 8 is a graph showing a time-course death curve of Larvae of Rhododendron

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location (C12N 1/20 C12R 1:07) (C12P 21/00 C12R 1:07) (72) Inventor Nobukazu Suzuki 5-6 Koyodai, Ryugasaki, Ibaraki Kubota Technology Development Laboratory Tsukuba Laboratory (72) Inventor Katsutoshi Ogihara 5-6 Koyodai, Ryugasaki City, Ibaraki Tsukuba Laboratory (72) Inventor Kazuki Atsushi 5-6 Koyodai, Ryugasaki, Ibaraki Kubota Technology Development Laboratory Tsukuba Laboratory (6) Inventor Hidetaka Hori 5-6 Koyodai, Ryugasaki, Ibaraki Kubota Technology Development Laboratory Tsukuba Research Co., Ltd. Indoor (72) Inventor Shoji Asano 5-6 Koyodai, Ryugasaki City, Ibaraki Prefecture Kubota Technology Development Laboratory Tsukuba Research The inner (72) inventor Kawasugi Tadaaki Ibaraki Prefecture Ryugasaki City Koyodai 5-6, Inc. KUBOTA Technology Development Institute, Tsukuba laboratory

Claims (2)

[Claims] 1. A Bacillus thuringiensis serovar japonensis belonging to Bacillus thuringiensis, which has the ability to produce an insecticidal toxin protein against larvae of Coleoptera insects.
Thuringiensis serovar japonensis strain buoy (Kikuyokojo No. 3465). 2. An insecticide containing as an active ingredient a toxin protein produced by Bacillus thuringiensis serovar japonensis strain buoy.