JP4768308B2 - Plant disease control microorganism and plant disease control method - Google Patents

Description

  The present invention relates to a technique related to control of plant diseases. More specifically, the present invention relates to a plant disease control technique using a specific Bacillus bacterium.

  In recent years, the biological control technology, also called “Biological agrochemicals”, has been actively promoted because of its low environmental impact and high safety against human livestock compared to plant disease control using chemically synthesized pesticides. The movement to use is active.

  This biological control technology uses pests that cause various diseases on plants and organisms that are natural enemies of microorganisms (insects, nematodes, bacteria, other microorganisms, etc.), substances produced by organisms (for example, antibiotics) ) And pheromones and other technologies that use physiologically active substances.

  Bacillus thuringiensis (abbreviated as Bt), which is widely used for pest control, is a representative example of the above-mentioned biological pesticides. The crystalline insecticidal protein (delta endotoxin) produced by culturing this Bt bacterium has already been put into practical use as a control for lepidopterous pests (for example, vegetable pests such as diamondback moth).

  In addition, Bacillus subtilis antagonizes certain pathogenic bacteria that cause plant diseases, so it has already been registered as a pesticide in Japan as a control agent for eggplant and tomato gray mold.

  In addition, some specific examples of prior art relating to plant disease control using bacteria belonging to the genus Bacillus are listed. Firstly, Patent Document 1 discloses culture of bacteria belonging to the genus Bacillus such as Bacillus subtilis. A disease control technique for agricultural and horticultural plants containing a spore fraction prepared so as to contain 50% by weight or more of spores in dry weight from a product is disclosed.

  Patent Document 2 discloses a bacterial body or culture of a Bacillus bacterium such as Bacillus licheniformis that has an antagonistic action on the microorganism in order to control a plant disease caused by the microorganism belonging to the genus Phytophthra. Discloses an epidemic control agent comprising an organic acid produced by the bacterium or a salt thereof as an active ingredient.

  Patent Document 3 discloses a plant disease control agent containing cells or cultures of Bacillus sphaericus.

  Patent Documents 4 and 5 disclose a plant disease control agent using a specific strain of Bacillus subtilis as an active ingredient.

JP-A-8-175919. Japanese Patent Laid-Open No. 2001-206811. JP2003-277210A. JP-A-6-133863. Japanese Patent Laid-Open No. 5-051305.

  The present invention has newly found a plant disease control function of a novel Bacillus bacterium, and thereby provides a plant disease control agent, a plant disease control medium using the Bacillus bacterium, and a plant disease control method of the present invention. The main technical issues.

The present invention finds 16S ribosomal DNA (16SrDNA) is Bacillus bacterium you exact and the nucleotide sequence shown in SEQ ID NO: 1, and operative according to the plant disease control Bacillus bacterium accession number FERM BP-10232 in new Based on
(1) The Bacillus bacterium, (2) Plant disease control agent using the Bacillus bacterium, (3) Plant disease control soil using the Bacillus bacterium, (4) Plant disease control method using the Bacillus bacterium ,I will provide a.

  The novel Bacillus bacterium according to the present invention is useful in plant disease control technology. In addition, the plant disease control agent according to the present invention is characterized by using the Bacillus bacterium, and the form and state of the drug can be determined as appropriate, or the method of use thereof is, for example, spraying, mixing, etc. Widely includes methods such as coating and dipping, and is not interpreted narrowly. The soil for controlling plant diseases according to the present invention is characterized by using the aforementioned Bacillus bacteria, and is used, for example, by a method of supplying an appropriate amount to soil for growing plants. The plant disease control method according to the present invention is characterized by using the aforementioned Bacillus genus bacteria, and the method is used so as to exert a plant disease control function with respect to the growth process and growth environment of the plant. The

  In the present invention, `` using a Bacillus bacterium '' means not only using the direct disease control action of the bacterium itself, which antagonizes in the living environment and the causative microorganism of the disease in relation to the plant disease for the purpose of control, Widely includes disease control by inducing disease resistance of plants.

Here, the Bacillus bacterium according to the present invention is a bacterium belonging to the genus Bacillus classified and specified by the base sequence of rDNA corresponding to the subunit of 16S as described above, and Bacillus sp. National Institute of Advanced industrial Science and technology Patent organism Depositary Center: is the accession number FERM bacteria of BP -10232, which have been deposited with the (address 1 central 6, Higashi, Tsukuba, Ibaraki 1-chome address 1).

The Bacillus bacterium according to the present invention has a completely identical base sequence to the 16S rDNA base sequence shown in SEQ ID NO: 1 .

  Currently, the phylogenetic classification method based on the base sequence of 16S rDNA is that 16S rDNA has an appropriate difference indicating the difference in species, and there is a sufficient amount of information to analogize the past evolution (mutation) process. It is a powerful technique for bacterial strain classification because it is easy to handle.

  The “plant disease” according to the present invention includes a wide range of diseases in which the Bacillus bacteria can be useful for disease control, and is not interpreted narrowly. Representative examples include powdery mildew, gray mold, plague, and rice seedling bacterial disease.

  By using the Bacillus genus bacterium according to the present invention in a plant growth environment, plant diseases can be effectively controlled. The culture soil for controlling plant diseases according to the present invention can suppress the growth of soil-damaging fungi that are harmful to the growth of plants, and can provide soil that hardly causes soil diseases to plants.

  Preparation of plant disease control agent (flowable agent).

  The bacterium belonging to the genus Bacillus according to the present invention has been confirmed to have a plant disease activity as a result of isolating microorganisms from natural soil and assaying the activity against plant diseases. Ribosomal DNA corresponding to the 16S subunit (Hereinafter, 16S rDNA) is a bacterium belonging to the genus Bacillus having the base sequence shown in SEQ ID NO: 1 in the attached sequence listing.

  The method and operation for obtaining the base sequence information of the rDNA are as follows. First, each specimen was inoculated into Nutrient Agar (Oxoid, Ebgland, UK) and cultured at 30 ° C. for 2 days, and then this specimen was used as a test specimen for DNA extraction. For the genome extraction, PrepMan Method (Applied Biosystems, CA, USA. Http://www.appliedbiosystems.co.jp/website/jp/home/index_g.jsp) was used. Using the extracted genomic DNA as a template, a 16S rDNA total base sequence of 1500 to 1600 bp was amplified by a known PCR method. Thereafter, the amplified 16S rDNA was sequenced to obtain 16S rDNA base sequence information (see Sequence Listing / SEQ ID NO: 1) of the sample. MicroSeqFull 16S DNA Bacterial Sequencing Kit (Applied Biosystems, CA, USA.) Was used for PCR product replication and cycle sequencing. GeneAMP PCR System 9600 (Applied Biosystems, CA, USA.) Was used for the thermal cycler, and ABI PRISM 3100 DNA Sequencer (Applied Biosystems, CA, USA.) Was used for the DNA sequencer. Used AutoAssembler 2.1 (Applied Biosystems, CA, USA.). The basic procedure from genomic DNA extraction to cycle sequencing was in accordance with Applied Biosystems protocol (P / N4308132 Rev. A).

In this way, Bacillus bacteria whose base sequence of 16SrDNA was identified and the taxonomic group was estimated were transferred to KABB medium (soy flour 10 g, sucrose 10 g, yeast extract 1 g, disodium hydrogen phosphate 1 g, starch 1 g. The mixture was cultured with shaking in water (1000 ml, pH 7) at 30 ° C. for 4 days to form spores, which were collected by centrifugation. Then, it suspended in water so that it might become a spore density | concentration of 1.0 * 10 < ¹¹ > cells / ml or more, and the target flowable agent was obtained. This flowable agent was used in Examples 3 to 5 described later.

The bacteria belonging to the genus Bacillus used in the present example are dated February 10, 2005 at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (address: 1st, 1st, 1st East, 1st Street, Tsukuba, Ibaraki Prefecture). in, in which it has been deposited as accession number FERM BP -10232.

  Examples that can be considered as related species of the genus Bacillus according to the present invention include Bacillus mojavensis, Bacillus atrophaeus, Bacillus amyloliquefaciens, Bacillus licheniformis, and the like.

  Production of soil for plant disease control.

In order to prepare culture soil containing Bacillus bacteria according to the present invention, first, 3 liters of cell seedling soil (cell culture soil 200 type: manufactured by Kureha Chemical) was sterilized by autoclave. This sterilized cell seedling soil was inoculated with one platinum ear of the Bacillus bacterium according to the present invention cultured on a potato dextrose agar medium. After inoculation, the cells were cultured at 28 ° C., and were grown until the bacterial concentration in the cell soil became 1 × 10 ⁷ cells / ml or more to obtain the target soil for controlling plant diseases. This plant disease control soil was used in Example 6 described later.

  Activity verification test for “tomato powdery mildew”.

  Cultivar Momotaro Eight as seedlings and Vespa as rootstock were sown on cell trays and cultivated for 25 days. These were grafted, planted in 3 inch pots and cultivated for 30 days to obtain seedlings having a growth stage of 7.5-8 leaves. When the seedlings were planted in an in-house greenhouse and grown for 3 months, when the growth stage reached the fourth fruiting stage, the flowable agent produced in Example 1 was diluted 2500 times and the tomato stems and leaves were prepared. Evenly sprayed on the part.

  In addition, as a comparative example for this example, a 1000-fold diluted solution of a commercially available microbial pesticide bottom killer wettable powder (registered trademark, manufactured by Idemitsu Kosan Co., Ltd.) was used and uniformly sprayed on the tomato leaves and leaves of the same tomato. In addition, a “control zone” in which no drug was sprayed was also prepared.

The size of the test group according to this example was 8 planting / 2.4 m ² / group, and each treatment was repeated 3 times. The amount of liquid at the time of spraying is a ratio of 350L / 10 are. Spraying was performed 5 times per week, and the activity against spontaneous tomato powdery mildew was investigated objectively. The disease area rate in the untreated area at the time of the survey was 12%. The results are shown in the following “Table 1”.

  The control value was calculated based on the following formula (the same applies to other examples).

  As can be seen from the results shown in the above-mentioned “Table 1”, the Examples have a control effect against tomato powdery mildew and have a higher control value than the comparative example in which the bottling hydrating agent is sprayed. Examples of powdery mildew that the Bacillus bacterium according to the present invention shows its control effect include, besides tomato powdery mildew, cucumber powdery mildew, melon powdery mildew, eggplant powdery mildew, and the like.

  Activity verification test for “cucumber gray mold”.

The flowable agent obtained in Example 1 was diluted with water to 1.7 × 10 ⁹ cells / ml, and this diluted solution was sprayed on the cucumber stems and leaves at the 0.2 leaf stage grown in the pot. . The amount of spray liquid is a ratio of 800 L / ha. After spraying, air-dried paper discs impregnated with a spore suspension of gray mold were placed on each of the five leaves of a cucumber strain. This was placed in a 20 ° C., high humidity sickness box, and after 3 days, the severity of the disease was examined. In addition, the diseased area ratio of the “control zone” where no drug was sprayed was 100%. The results are shown in the following “Table 2”.

  As can be seen from the results shown in the above-mentioned “Table 2”, the example which is a flowable agent sprayed area showed a high control effect against cucumber gray mold.

  Activity verification test for “tomato gray mold”.

The flowable agent obtained in Example 1 above was diluted with water so as to be 1.6 × 10 ⁹ cells / ml, and 40 ml with respect to the foliage part of the tomato grown in the pot until the second stage fruiting stage. / Sprayed at a pot ratio.

On the 4th day after spraying, a spore suspension of gray mold (spore concentration: 1.0 × 10 ⁵ cells / ml) was sprayed onto the tomato stems and leaves. The amount of spray inoculation is 330 ml / m ² . After that, they were put into a high-humidity box for 3 days to cause illness, and the degree of illness was investigated objectively. The disease area ratio in the non-sprayed area was 76%. The results are shown in the following “Table 3”.

  As can be seen from the results shown in the above-mentioned “Table 3”, this example, which is a flowable agent spraying area, had a control effect against tomato gray mold. The gray mold that the Bacillus bacterium according to the present invention exhibits its control effect is, for example, gray for eggplant, peppers, strawberries, grapes, etc. in addition to the cucumber (see Example 4) and tomato (see Example 5). Mention of mold disease.

  Activity verification test against "rice seedling blight disease".

The flowable agent prepared in Example 1 was diluted with water to 6.5 × 10 ⁷ cells / ml, and 20 seeds of rice seedlings (variety: Nipponbare) suffering from rice seedling bacterial disease were added to this diluted solution. The immersion treatment was conducted at 24 ° C. for 24 hours. Thereafter, seeding was carried out at 20 ° C. for 4 days, and sprouting treatment was performed at 32 ° C. for 24 hours.

  The seeds thus obtained were sown in a 10 cm × 15 cm plastic cup packed with a commercially available granular rice soil (manufactured by Kureha Chemical Industry Co., Ltd.), covered with soil, put in a warm nursery device and germinated ( Temperature condition: 32 ° C., 2 days). After taking out from the seedling raising device, it was cultivated in a glass greenhouse, and the severity of the disease was investigated objectively 21 days after sowing. The diseased seedling rate in the control group (the flowable agent-free group) was 30%. The results are shown in the following “Table 4”.

  As can be seen from the results shown in the above-mentioned “Table 4”, in the example where the flowable agent was used, a high control effect against the rice seedling bacterial disease was shown.

  Activity verification test against "tomato plague".

  Cell seedling soil (cell culture soil 200 type: Kureha Chemical Co., Ltd.) and field soil were mixed at a volume ratio of 1: 1, and the stem and leaf of a tomato affected by the plague were mixed therein to prepare a diseased soil. To this diseased soil, the plant disease control soil obtained in Example 2 was mixed at a volume ratio of 10%, and placed under cultivation management at 20 ° C. for 1 day.

  Tomato seedlings (variety: Fukuju, 2.5 leaf stage) were transplanted to this, and the cultivation was managed in a wet room and the disease was promoted. The survey was conducted objectively 5 days after transplantation. The disease area ratio in the untreated area was 46%. The results are shown in the following “Table 5”.

  As can be seen from the results shown in the above-mentioned “Table 5”, this example, which is a flowable agent spraying area, had a control effect against tomato plague. Examples of the plague that the Bacillus bacterium according to the present invention exhibits its controlling effect include, for example, cucumber, watermelon, potato, melon, and pumpkin.

  As is apparent from the results of the above examples, the Bacillus bacterium according to the present invention has a plant disease control effect.

  INDUSTRIAL APPLICABILITY The present invention can be used as a plant disease control technique using a Bacillus bacterium, a so-called biological pesticide. For example, it can be used as a plant disease control agent, a plant disease control soil, and a plant disease control method.

Claims (5)

A bacterium belonging to the genus Bacillus having 16S ribosomal DNA (16SrDNA) completely matching the nucleotide sequence shown in SEQ ID NO: 1 and having a plant disease control activity . A plant disease control agent using a Bacillus bacterium in which 16S ribosomal DNA (16SrDNA) completely matches the nucleotide sequence shown in SEQ ID NO: 1. A soil for controlling plant diseases using a Bacillus bacterium in which 16S ribosomal DNA (16SrDNA) completely matches the nucleotide sequence shown in SEQ ID NO: 1. A method for controlling plant diseases using Bacillus bacteria,
The method for controlling plant diseases, wherein the Bacillus bacterium is a Bacillus bacterium in which 16S ribosomal DNA (16SrDNA) completely matches the base sequence represented by SEQ ID NO: 1. Bacillus genus bacteria of accession number FERM BP-10232.