JP3251137B2 - Bacillus thuringiensis new strain - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel Bacillus thuringiensis isolate having insecticidal activity against dipteran pests, especially Drosophila.

[0002]

2. Description of the Related Art Bacillus thuringiensis (BT), which is most widely used as a microbial insecticide instead of a chemical insecticide, forms a crystalline toxin protein during the sporulation stage, and this toxin protein is It is known that pests that have ingested died after rupture of the intestinal tract and the like.

[0003] BT has a different insecticidal activity depending on its subspecies. For example, subspecies Krusutaki and Aisawai have
Toxic to Lepidoptera and Diptera insects (Dulmage, HT: J. In
vertebr. Pathol., 15, 232 (1970)), Islaelensis is a dipteran insect (LJ Goldberg: Mosq. News, 3
7, 355 (1977)) and Tenebrionis are known to be toxic to Coleoptera beetles, respectively (Japanese Patent Application Laid-Open No.
0-156382). However, microbial insecticides have a narrower range of application of pests than chemical pesticides, so in order to extend the range of application, several types of BT may be mixed and used, or the range of application may be reduced by genetic engineering techniques. Methods such as spreading are taken.

[0004] On the other hand, however, the use of BT insecticides with a wide range of applications, or the use of several BT insecticides,
It will lead to the same harm as chemical pesticides. In other words, it has activity against insects and parasites that are beneficial due to its wide host range, and there is a risk of destroying the biological control action of the natural environment. In particular, the unpleasant sanitary pests generated in large quantities in simple household septic tanks and sewage treatment tanks consist of almost uniform species depending on their growing environmental conditions.
The use of the T insecticide is more suitable in consideration of environmental safety. For example, there is a report that uses Israelensis to control flies generated in sewage filtration tanks (J.
Houston: Wat. Res. 23, 379, (1989)), and its applicable pests are Limnophyes minimus and Metoriocnemus hygropetricu.
s, Psychoda severini, Psychoda alternata, Sylvico
La fenestralis, Orthocladius fuscimanus and widely, exhibit insecticidal activity to many dipteran insects. This report includes the report of the unpleasant sanitary insect pest Drosophila (Telmatoscopus albipunct), which specifically occurs in simple household septic tanks and sewage treatment tanks.
atus), there has been no report of a BT insecticide exhibiting pesticidal activity specific to so far.

[0005]

SUMMARY OF THE INVENTION It is an object of the present invention to solve the above-mentioned problems by using the fruit fly (Telmatoscopus
albipunctatus), a novel Bacillus thuringiensis showing pesticidal activity
It is an object of the present invention to obtain an isolate, and to provide an insecticide or an insecticide method which is effective for controlling Drosophila using the isolate.

[0006]

Means for Solving the Problems The present inventor has proposed that Bacillus thuringiens having insecticidal activity against Drosophila in particular among dipteran pests.
sis) This problem was solved by successfully isolating a new strain. This new isolate, designated by the present inventors as 88-KO-14-45, has been deposited with the National Institute of Bioscience and Human Technology, under the deposit number FERM P-14496 and has the following properties: Have.

(1) This novel Bacillus thuringiensis isolate 88-KO-14
-45 was isolated from the soil in Katsurahama, Kochi Prefecture. (2) The isolated new strain is a normal agar medium (pH 7.
6) Form large rough white colonies on top. (3) This bacterium is a gram-positive rod and has flagella. (4) Bacillus thurin
giensis) is classified according to its flagellar serotype, and the isolate of the present invention is classified into H antigen type 33, Serovar leesis. Fungi of this serotype have so far not shown any insecticidal activity against any insects. The activity of the isolate 88-KO-14-45 of the present invention on Drosophila melanogaster is the first report in Bacillus thuringiensis classified into this serotype and showing insecticidal activity. (5) The new strain of the present invention is oxygen demanding, that is, aerobic.

[0008] The novel Bacillus thuringiensis isolate 88-KO-14 of the present invention.
-45 is an unpleasant hygiene pest, Drosophila (Telmatoscop)
us albipunctatus) show strong insecticidal activity against larvae. However, the same dipteran insect, Culex pipiens m
olestus), Anopheles stephensi, Anthracidae (Orthocladius akamusi), Housefly (Musuca
domestica), the new strain of the present invention shows no toxicity.
The property of exhibiting strong toxicity only to Drosophila but not other insects is particularly effective in consideration of environmental safety.

Thus, according to the present invention, there is provided an environmentally friendly insecticide for Drosophila containing a culture of a new strain of Bacillus thuringiensis 88-KO-14-45 as an active ingredient. The culture of the new strain 88-KO-14-45 can be performed using a standard medium. For example, when cultured at 30 ° C. using an agar medium, spores are formed in 3 to 5 days, and a crystalline toxin protein is produced. The spores of the novel isolate 88-KO-14-45 and the crystalline toxin protein as active ingredients can be recovered by scraping off the agar medium. Further, the novel isolate of the present invention can be cultured using an appropriate liquid medium, in which case,
The active ingredient can be recovered by centrifugation or filtration after the culture.

The novel isolate 88-KO-14-4 of the present invention
The crystalline toxin protein produced during the sporulation stage by 5 was 3% when measured by electrophoresis (SDS-PAGE).
It has been found that the molecular weight is such that it has a strong line near 1,62,66,72, and 78 kilodaltons and a weaker line near 55 kilodaltons.

The insecticide of the present invention is a novel isolated strain 8
8-KO-14-45 (spores) or a crystalline toxin protein is provided as a mixture obtained by suspending in water or adding a suitable known compound, and feeding the mixture to a target pest. To exterminate. The concentration of the active ingredient in the pesticide mixture is generally between 1 × 10 ^{8 and} 10 ¹⁰ / ml as spore concentration.

[0012]

[Example 1]

[New Bacillus thuringiensis isolate 88-KO
Isolation of 14-45] 0.5 g of Katsurahama soil in Kochi prefecture was weighed into a test tube containing 4.5 ml of sterilized water, and sufficiently suspended. Next, this soil suspension (10 ^-1 diluted solution) was left in a water bath at 70 ° C. for 10 minutes to perform heat treatment. Next, for further dilution, 0.5 ml of the above-mentioned soil suspension was taken and added to 4.5 ml of sterilized water to prepare a 10 ^-2 diluted solution. In the same manner, a serially diluted solution was sequentially prepared up to a 10 ^-5 diluted solution.

Next, this diluted sample was added to a normal agar medium (meat extract 10 g, polypeptone 10 g, sodium chloride 2 g,
20 ml of agar, 1 liter of water, pH 7.6) 0.1 ml
The cells were smeared and cultured at 28 ° C. for 3 to 5 days. Colonies that grew after culturing for a predetermined period were observed under a phase-contrast microscope, and those in which spores observed in white and crystalline proteins observed in black were observed in the same cell were selected.

Example 2 [New Bacillus thuringiensis isolate 88-KO]
Identification of serotype of -14-45] The serotype was identified by the method of Ohba et al. (Ohba, M .: J. Invertebr. Pathol. 32, 303-309).
(1978)). New Bacillus thuringiens of the present invention
is isolate 88-KO-14-45 in an L-shaped test tube
of medium (10 g of meat extract, 10 g of polypeptone, 2 g of sodium chloride, 1 liter of water, pH 7.6) and gently shaken at 37 ° C. for 3 to 4 hours. 3-4
After an hour, one drop of this suspension was placed on a glass slide for 20 minutes.
It was mixed with one drop of serum (Nos. 1-34) diluted 〜50-fold. After 3 to 5 minutes, an agglutination reaction was determined, and a reaction was observed only with the serotype 33 serum. Thus, serotype 33 lysis was determined.

[Example 3] [Toxicity test on dipteran insects] The novel Bacillus thuringiensis isolate 88-KO-14-45 of the present invention was cultured on a normal agar medium at 28 ° C for 5 days and then collected. Washed twice or three times with distilled water. Drosophila (Telmatoscop
us albipunctatus), Culex pipiensmolest (Culex pipiensmolest)
us), Anopheles stephensi and Orthocladius akamusi have a spore concentration of 1
The 0 ⁸ / ml and made as culture was suspended in distilled water, placed in a 5 larvae each insect therein were tested for 3 days at 25 ° C. For the housefly (Musuca domestica), the culture was mixed in the feed so that each larva feeds on 10 ⁸ spores, and 2 larvae were similarly fed to 5 larvae.
Tested at 5 ° C for 3 days. One or more crystalline proteins are formed from one cell during spore formation.

The results of the toxicity test are shown in Table 1. For the active fruit fly, 20 larvae were put into a culture suspension diluted to various concentrations, the number of dead insects after 24 hours was measured, and the LC50 was determined by the probit method.
As a result, the LC50 value was 217 μg wet weigt / ml. According to this example, the novel isolate 88-KO-1 of the present invention was obtained.
4-45 was found to have insecticidal activity specifically to Drosophila among diptera insects.

[0017]

[Table 1]

[Example 4] [Purification of the crystalline toxin protein produced by the novel isolate 88-KO-14-45] The novel isolate 88-KO- of the present invention
After culturing 14-45 in a normal agar medium (meat extract 10 g, polypeptone 10 g, sodium chloride 2 g, agar 20 g, water 1 liter, pH 7.6) at 27 ° C. for 5 days, cells are collected and washed with a 1 M sodium chloride solution. And centrifugation three times. Partial purification of the crystalline toxin protein was performed by a two-phase separation method (Goodm
an. NS: J. Bact. 94, 485 (1967)).
The lower layer containing the crystalline toxin protein was diluted with water and collected by centrifugation. Sucrose density gradient centrifugation was performed in order to purify further until it became pure.

70 W / V%, 80 W / V%, 90 W / V
% Sucrose solution was layered on a 10 ml centrifuge tube, and 0.5 ml of the partially purified crystalline toxin protein was further layered on the top. After centrifugation at 22,000 rpm for 2 hours, 70 W
/ V% -80 W / V% The pure purified crystalline toxin protein that precipitated at the interface was recovered. When this crystalline toxin protein was subjected to SDS-PAGE, 31,62,
Lines appeared at around 66, 72, and 78 kilodaltons, and weak lines around 55 kilodaltons.

[0020]

The novel isolate 88-KO-14 according to the present invention
45 is an unpleasant sanitary insect pest, Drosophila melanogaster (Telmatoscopus albipun), which is generated in household simple septic tanks and sewage treatment tanks.
ctatus), and has a pesticidal activity, and can be effectively used to control this pest.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ identification code FI C12R 1:07) C12R 1:07) (58) Investigated field (Int.Cl. ⁷ , DB name) C12N 1/20-1 / 21 BIOSIS (DIALOG) CA (STN) JICST file (JOIS) WPI (DIALOG)

Claims (2)

(57) [Claims] 1. A new strain of Bacillus thuringiensis 88- having specific insecticidal activity against Drosophila melanogaster.
KO-14-45 (Biotechnological Research Institute Accession No. F
ERM P-14496). 2. A new strain of Bacillus thuringiensis 8
An insecticide for Drosophila containing a culture of 8-KO-14-45 (Accession No. FERM P-14496, Institute of Biotechnology and Industrial Technology) as an active ingredient.