JP5773411B2 - Novel microorganism and microbial pesticide using the same - Google Patents

Description

  The present invention relates to a novel microorganism having an excellent pest control effect, and a microbial pesticide using the same.

  Conventionally, as an important means for controlling pests, chemically synthesized insecticides such as organophosphorus insecticides that can provide remarkable effects at low cost have been widely used. However, chemical synthetic pesticides have problems such as environmental persistence, toxicity to non-target organisms, and the development of pesticide-resistant pests.

  Therefore, as an alternative to these chemically synthesized insecticides, biological pest control technologies such as pheromone attractants and biological pesticides that apply the inherent properties of living organisms are being developed, and next-generation pest control with a low environmental impact is being promoted. Expected as a technology.

  Pest control technology using a pheromone attractant is a technique that prevents spawning on crops by disrupting male and female communication by suspending insect sex pheromone on the ceiling of a greenhouse, etc., and has strong species specificity Therefore, it is possible to ensure high safety against non-target organisms. However, on the other hand, it is difficult to control multiple crop pest groups, and it is necessary to predict the type of pests that occur well before the time of the damage and to install a pheromone attractant accordingly. There are disadvantages.

  On the other hand, pest control technology using biological pesticides is a technique for killing or suppressing behavior by applying natural enemy organisms against pests and crop pathogenic microorganisms, and insects and nematodes as the natural enemy organisms. The method is roughly divided into the method using microorganisms and the method using microorganisms.

Among these methods, insects and nematodes used as natural enemies include, for example, a method of applying insects such as the coleman abalone that prey on crop pests in a greenhouse, a steiner nematode that is parasitic on insects or exhibits pathogenicity. A method of applying a nematode such as Graserai ( Steinernema glaseri ) is known. However, these methods have a disadvantage that a stable effect is difficult to obtain because the insecticidal effect is exhibited by predation or parasitism of the pest by natural enemy organisms.

  As described above, biological insecticides that use pheromone attractants and insects and nematodes are difficult to achieve a stable pest control effect. ) Is attracting attention. Microbial pesticides have advantages in that they are highly safe against non-target organisms including humans and the environment and have long-lasting effects compared to chemically synthesized insecticides. In addition, unlike pheromone attractants, it can be applied depending on the state of pest occurrence, and since microorganisms grow faster than insects and nematodes, it is considered that the productivity of agricultural chemicals is high.

By the way, many microbial pesticides currently registered in Japan target crop pathogenic microorganisms, and a few pesticidal pesticides target pests. Among them, typical microbial pesticides for insect pests are BT agents using an entomopathogenic bacterium, Bacillus thuringiensis , especially fly eyes, butterfly eyes, beetles, etc. (See, for example, Patent Document 1). Other pesticidal pesticides that use pest control are Verticillium lecanii ( Zimm. ) Viegas and Beauveria bassiana Vuillemin. It is mainly used to control aphids.

JP 2010-001237 JP

  However, in recent years, there have been reports of the appearance of pests that are resistant to BT agents, and the microbial pesticides using the above-mentioned filamentous fungi have a problem that insects that can be controlled are limited. For these reasons, improvement of microbial pesticides for the purpose of pest control is now required, and as one of the improvement measures, search for entomopathogenic microorganisms that can be used as new microbial pesticides is required.

  The present invention has been made in view of the above problems, and an object thereof is to provide a novel microorganism that can be used as a microbial pesticide for pests, and a microbial pesticide using the same. .

  As a result of intensive studies to solve the above problems, the present inventors have found that a novel strain isolated from larvae of the carnivorous insect, Crocopus quercivorus, has a pest control effect and completes the present invention. It came to.

That is, the present invention provides a novel microorganism, Bacillus subtilis An11-1 strain (NITE P-997).

Moreover, this invention provides the microbial pesticide containing the Bacillus subtilis ( Bacillus subtilis ) An11-1 strain (NITE P-997) as an active ingredient.

  As described above, according to the present invention, a novel microorganism having an excellent control effect against pests and a microbial pesticide using the microorganism can be provided.

Molecular phylogenetic tree of microorganisms according to the present invention prepared by a proximity bond method. Molecular phylogenetic tree of microorganisms according to the present invention prepared by the maximum likelihood method. The graph which shows the insecticidal effect to Spodoptera litura by the microorganisms of this invention. The graph which shows the insecticidal effect to Spodoptera litura by the microorganisms of this invention and the standard strain of Bacillus subtilis. The table | surface which shows the insecticidal effect to the mosquito by the various strains of the microorganisms of the present invention and Bacillus subtilis.

  Hereinafter, modes for carrying out the present invention will be described.

  The microorganism according to the present invention is a strain obtained as a result of selection by an insecticidal activity test against a moth Lepidoptera which is a lepidopteran pest, from bacteria collected from a larva of a carnivorous insect, Crocopus quercius.

  It is known that a crocodile moth larva known as Aridigok injects exhalation solution containing digestive enzymes and paralytic components into its body when it prey on insects, and paralyzes the insects to be eaten. At this time, the blackened phenomenon observed when the insect pathogenic microorganisms are infected is observed in the insects injected with the repellent solution. Based on this, the present inventor predicts that an entomopathogenic microorganism group and its toxin are present in the gastrointestinal tract of the crocodile moth larvae, and that this is involved in the paralysis of insects that prey, and The bacteria inside were isolated and cultured. In order to search for microorganisms that have an insecticidal effect against the larvae of Lepidoptera insects that account for a relatively high proportion of the agricultural pests among the isolated strains, artificial diets containing each strain were prepared and Lepidoptera We tried oral administration to the third-instar larvae of the insect, Spodoptera litura. As a result, one strain showing a remarkable insecticidal activity was isolated and named An11-1 strain.

The mycological properties of the An11-1 strain are as follows. The bacteriological properties were tested based on the NCIMB test method and the following reports 1-3.
Previous report 1: Barrow GI and Feltham RKA, Cowan and Steel's Manual for the identification of Medical Bacteria. 3rd edition, 1993, Cambridge University Press
Previously reported: Toshikazu Sakazaki, Goro Yoshizaki, Kanji Miki: New Bacteriological Studies Course, 2nd edition, 1988, Kinkadai Publishing, Tokyo Previously published 3: edited by Takeharu Hasegawa, Classification and identification of microorganisms (2), 1995 , Academic Publishing Center

The results of the molecular phylogenetic tree analysis for the An11-1 strain are shown in FIGS. Fig. 1 shows the phylogenetic tree created by the proximity coupling method (MEGA ver. 4.0, base substitution model: Tamura-Nei distance, calculation of gamma correction distance: DAMBE ver. 5.2.5), and Fig. 2 shows the maximum likelihood method. (GARLI ver. 0.96, calculation of consensus tree: Phyutility ver. 2.2) shows the phylogenetic tree created. Molecular phylogenetic analysis is based on a report by Rooney et al. (Rooney, AP, et al., Int. J. Syst. Evol. Microbiol., 2009, 59, 2429-2436.), And six housekeeping genes (DNA gyrase α -subunit gene ( gyrA ), RNA polymerase β-subunit gene ( rpoB ), phosphoribosylaminoimidazole carboxamide formyltransferase gene ( purH ), DNA polymerase III α-subunit gene ( polC ), 60 kDa heat-shock protein groEL gene ( groEL ), 16S rRNA gene ( 16S rRNA )) partial sequences ( gyrA 928 bp, rpoB 964 bp, purH 875 bp, polC 777 bp, groEL 835 bp, 16S rRNA 1169 bp). BLAST homology search was performed with Apollon 2.0 (Techno Suruga Lab, Shizuoka), and the international base sequence database GenBank / DDBJ / EMBL was used as the database. As a result, the An11-1 strain was considered to belong to Bacillus subtilis .

Therefore, a DNA-DNA hybrid formation test was performed between the An-11 strain and a Bacillus subtilis reference strain ( B. subtilis NBRC13719). As a result, as shown in the following table, the An11-1 strain and the B. subtilis NBRC13719 strain showed 79% homology with each other. It should be noted that strains showing homology of 70% or more in the DNA-DNA hybridization test are considered to be the same species.

From the results of the above mycological properties, molecular phylogenetic tree analysis, and DNA-DNA hybridization test, the An11-1 strain was identified as a microorganism belonging to Bacillus subtilis . However, since an insecticidal activity against Spodoptera litura was not observed in the standard strain of Bacillus subtilis ( Bacillus subtilis 168trpC2), the An11-1 strain was judged to be a new strain (see [Test Example 2] described later). This strain is deposited under the accession number NITE P-997 at the Patent Microorganism Deposit Center, National Institute of Technology and Evaluation.

  The microbial pesticide (pest control agent) according to the present invention contains the above strain as an effective bacterium.

  The strain is preferably prepared by using a liquid medium and performing shaking culture at 20 to 50 ° C. and pH 5 to 9 for 24 hours or more, but is not limited thereto. Alternatively, it may be adjusted by culturing using a solid medium. The type of the medium is not particularly limited as long as the bacteria can grow. For example, an SCD liquid medium (soybean / casein / digest / broth medium) can be preferably used.

  The microbial pesticide according to the present invention may be used in the form of a microbial cell containing the above microbial cell or a microbial cell excluding the cultivated cell, but may be held on a carrier made of a predetermined solid, granule, powder, etc. It is desirable to use the dosage form. Moreover, it is good also as a liquid formulation by disperse | distributing to a predetermined liquid. In addition, when a microbial cell is prepared by liquid culture, this microbial cell is collect | recovered by centrifugation or filtration, and when adjusted by solid culture, this microbial cell is collect | recovered by scraping off from a culture medium.

  The above-mentioned microbial pesticide can be exerted by spraying it on the pest habitat and feeding the pest. In addition, the amount used is appropriately adjusted according to the type of pests to be controlled, the place and period of use, the degree of damage, and the like.

  The microbial pesticide according to the present invention can be suitably used for controlling various insect pests, particularly insects including butterfly insects and fly insects. Examples of the Lepidoptera insects include moths and butterflies such as weevil such as Lotus moth, and fly insects include, for example, mosquitoes, but are not limited thereto. Absent.

[Test Example 1]
An artificial bait containing the microorganism according to the present invention ( Bacillus subtilis An11-1 strain) was prepared, and this was orally administered to the third-instar larvae of the moth Lepidoptera, which was evaluated for insecticidal activity.

Specifically, first, 100 mL of an SCD liquid medium is added to a 500 mL Erlenmeyer flask, and the microorganism according to the present invention is added thereto, followed by shaking culture at pH 6 and 37 ° C., and after 48 hours from the start of the culture. A part of was collected. The collected culture solution is adjusted so that the number of viable cells becomes 10 ⁸ cells / mL, and the adjusted culture solution is added to 500 μL of dry yeast 60 mg, sodium alginate 10 mg, sucrose 20 mg, sodium ascorbate 4 mg. After adding a 300 μL aqueous solution containing 1%, 4 μm CaCl ₂ 200 μL was added and mixed and solidified, and this was used as one artificial bait.

  Two artificial baits prepared as described above (corresponding to the microbial pesticide in the present invention) were placed in a 9 cm petri dish, and 10 third-instar larvae fasted for 24 hours were placed there and allowed to stand at 25 ° C. for 5 days. The insecticidal effect by food was evaluated. As controls, an artificial bait prepared using 500 μL of sterile distilled water instead of the above bacterial solution and an artificial bait prepared using 500 μL of SCD liquid medium instead of the above bacterial solution were prepared in the same manner as described above. Then, it was orally administered to 10 3rd instar larvae of Spodoptera.

  As a result of the above, as shown in FIG. 3, the section (Comparative Example 1) fed with artificial food prepared using sterilized distilled water instead of the bacterial solution, and the artificial material prepared using the SCD liquid medium instead of the bacterial solution In the group fed with food (Comparative Example 2), the mortality rate of the larvae on the fifth day after administration of the artificial food was 5% or less, whereas the group fed with the artificial food containing the microorganism according to the present invention In Example 1, the mortality rate on the first day after administration of the artificial diet was about 15%, and the mortality rate on the fifth day after administration was about 90% (Note that the graph in FIG. 3 is the same test). Is the average of the results obtained by performing 3 times). Thereby, it was confirmed that the microorganisms according to the present invention have a remarkable insecticidal effect against Spodoptera litura.

[Test Example 2]
In the same manner as in Test Example 1, an artificial bait containing the microorganism according to the present invention, an artificial bait containing a standard strain of Bacillus subtilis ( Bacillus subtilis 168trpC2), and sterilized distilled water instead of the above bacterial solution An artificial bait was prepared and was orally administered to 10 third-instar larvae of Spodoptera litura in the same manner as above.

  As a result, as shown in FIG. 4, in the group (Example 2) to which the artificial bait containing the microorganisms according to the present invention was administered, the larval mortality on the 6th day after administration was about 85%, and a high insecticidal effect was observed. On the other hand, in the group (Comparative Example 4) to which the artificial bait containing the standard strain of Bacillus subtilis was administered, the mortality of the larvae on the 6th day after the administration was administered with the artificial bait using sterile distilled water. And the insecticidal effect was not recognized (the graph of FIG. 4 shows the average of the results obtained by performing the same test three times).

[Test Example 3]
Culture medium containing a microorganism according to the present invention was cultured in the same manner as in Test Example 1, the culture solution containing the standard strain of Bacillus subtilis subtilis, culture medium containing standard strains of Bacillus subtilis spizizenii, and Bacillus subtilis inaquosorum A culture solution containing a standard strain was prepared, and a culture solution adjusted to 10 ⁸ cells / mL was added to water containing 10 larvae of the fly mosquito, a fly insect, so that the turbidity was 0.03. . As a result, a result as shown in FIG. 5 was obtained. Specifically, in the culture solution containing the microorganism according to the present invention, a high insecticidal effect of 80% or more against Culex pipiens was observed, whereas in the culture solution containing each of the above standard strains, Bacillus subtilis subtilis However, 20%, Bacillus subtilis spizizenii and Bacillus subtilis inaquosorum showed almost no insecticidal effect at 0%.

Claims (2)

Bacillus subtilis An11-1 strain (NITE P-997). A microbial pesticide containing Bacillus subtilis An11-1 strain (NITE P-997) as an active ingredient.

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