JP2013042690A - New microorganism and plant disease prevention material using the new microorganism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new microorganism against fruit and vegetable diseases, and a plant disease prevention material using the microorganism.SOLUTION: Provided are Bacillus amyloliquefaciens S13-3 strain isolated from the natural environment, and the plant disease prevention material using the strain. The microorganism is effective for preventing a wide range of plant diseases caused by filamentous fungi and exhibits antimicrobial effect owing to its externally produced antibiotic substance. It can be used in combination with other agrochemicals and is useful for reduced pesticide cultivation or organic farming cultivation.

Description

The present invention relates to a new species of Bacillus amyloliquefaciens, and a plant disease control material using cells and cultures of the microorganism.

The bio-pesticide market is only about 800 million yen (FY2009), and the scale of the pesticide market is less than 1%. However, biological pesticides and biomaterials have been affected by the increase in the number of producers trying organic and reduced pesticide cultivation, the recent improvement in consumer awareness of pesticide residues, and the enforcement of the revised Food Sanitation Law, which enforced regulations on pesticide residues. The attention to is expected to increase further in the future. The crop-reducing pesticide cultivation and the accompanying image of “safety and security” are very effective in marketing strategy. In addition, the revision of the Agricultural Chemicals Control Law and the introduction of a residual pesticide positive list system have greatly boosted the market for biological pesticides.
Among biological pesticides, microbial pesticides using microorganisms are attracting attention.
At present, four microbial pesticides are registered for soft rot, gray mold, etc., and it is believed that they compete against pathogens for their homes and nutrients, and produce or parasitize antagonists. Compared with chemical pesticides, microbial pesticides use (1) microorganisms that survive in the natural world, so the reaction is mild and the burden on the human body and the environment is extremely low. There are significant advantages such as clearness and prevention of multiple spraying, and (3) long-lasting effects are expected if microorganisms settle in plants.
Conventionally, as a biological pesticide using Bacillus subtilis, for example, a plant disease control containing a spore fraction adjusted to contain 50% by weight or more of dry spores from a culture of Bacillus bacteria such as Bacillus subtilis The method is known. Bacillus licheniformis and Bacillus amyloliquefaciens (Patent Documents 1 to 4), which are known as other antagonistic bacteria of the genus Bacillus, cause phytophytra and mulberry white rot, which are well-known as potato epidemics Effective against pathogens of the genus Roselinia.
However, the above-mentioned conventional plant disease control agent has a considerably lower control effect than chemical pesticides, and has a difficult problem such as a narrow range of adaptation to pathogenic bacteria and only a specific control effect.

Patent Document 1: JP-A-11-246324 JP 2002-145712 A JP-A-63-38482 JP2009-101063 International Publication Number WO2010 / 004713A1

Therefore, the problem to be solved by the present invention is that not only a preventive effect but also a therapeutic effect can be expected, and a novel microorganism applied to various plant pathogens and a plant disease control using this novel microorganism To provide materials.

In order to solve the above problems, a novel microorganism according to claim 1 of the present invention is Bacillus amyloliquefaciens S13-3 (accession number: NITE P-1121). It is what.

A plant disease control material according to claim 2 is characterized in that it contains a culture of the Bacillus amyloliquefaciens S13-3 strain and / or a microbial cell as an active ingredient.

The plant disease control material according to claim 3 is characterized in that it effectively acts on late grape rot, gray mold disease, and strawberry anthracnose.

Bacillus amyloliquefaciens S13-3 strain, Bacillus amyloliquefaciens S13-3 strain culture according to the present invention, and / or microbial cells of Bacillus amyloliquefaciens S13-3 strain as active ingredients As a plant disease control material, the preventive effect and the suppressive effect on a wide variety of plant pathogens such as anthrax, late rot, gray mold, and black spot fungus can be expected.

The phase-contrast micrograph of the observation result of the microbial cell and spore of Bacillus amyloliquefaciens S13-3 strain of this invention. The photograph of the spraying test result in the field with respect to the anthracnose fungus of the strawberry of Bacillus amyloliquefaciens S13-3 of this invention (upper figure: untreated area, lower figure: S13-3 stock area). Results of disease incidence (%) between Bacillus amyloliquefaciens S13-3 strain and control material in trials using strawberry cut leaves. Scattering test of S13-3 strain using strawberry cut leaves, photograph of degree of disease (upper figure: no treatment, lower figure: S13-3 strain). Antibiotic secretion test and anti-culture results of Bacillus amyloliquefaciens S13-3 strain. The microscope picture of the mycelia tip of grape late rot fungus when it was countercultured with the Bacillus amyloliquefaciens S13-3 strain of the present invention. The growth inhibition photograph of the grape late rot fungus by the antibiotics which Bacillus amyloliquefaciens S13-3 strain | stump | stock of this invention produces.

Hereinafter, the present invention will be described in detail. The Bacillus amyloliquefaciens S13-3 strain according to the present invention is selected from thousands of bacteria isolated from the soil of the Botanical Research Institute (Ainohara Town, Atami City, Shizuoka Prefecture) owned by the New Technology Development Foundation. It is an identified bacterium and has a characteristic of showing a strong inhibitory effect against various pathogenic bacteria. The microbiological characteristics and various properties of the Bacillus amyloliquefaciens strain S13-3 are shown below.

A. Bacterial form
(1) Shape: Chosen
(2) Size: 0.8 × 9μm
(3) Mobility: Yes

B. Colony morphology (31 ° C, 1 day culture)
(1) Medium name: SCD agar medium
(2) Colony size: 9mm
(3) Colony shape: irregular circle
(4) Colony uplift: flat
(5) Shape around the colony: wavy
(6) Colony surface shape: wrinkled
(7) Colony quality: sticky
(8) Colony transparency: opaque
(9) Glossy colony: dull light
(10) Colony color: light beige

C. Physiological properties

The base sequence of 16S rDNA is shown in the following SEQ ID NO.

Sequence number

1509 bp

Based on the above microbiological properties and the sequence number of the base sequence of 16S rDNA, this strain was identified as Bacillus amyloliquefaciens. Bacillus amyloliquefaciens S13-3 On August 9th, it was deposited with the independent evaluation corporation Product Evaluation Technology Climate Patent Microorganism Deposit Center (2-5-8 Kazusa-Kamashita, Kisarazu City, Chiba Prefecture) and was deposited under the deposit number “NITE P-1121”.

The Bacillus amyloliquefaciens strain S13-3 according to the present invention is not limited to the base sequence that completely matches the 16S rDNA base sequence shown in the above SEQ ID NO. Even in the case of consisting of a base sequence in which one or several bases are deleted, substituted or added or inserted, in plant disease control action, particularly grape disease or strawberry disease control action, Bacillus amyloliquefaciens S13-3 It shall have the same character.

The medium that can be used in the present invention is not particularly limited as long as the strain can be grown by culture. For example, glucose, sucrose, yeast extract, meat extract, polypeptone, peptone, etc. are used as the carbon source. In addition, sodium, potassium, magnesium, iron, calcium and the like are added as other nutrients.

The culture conditions in the present invention can be cultured under aerobic conditions, for example, by aeration stirring, shaking culture, or a solid medium method. The culture conditions are not particularly limited, but a culture temperature of 30-37 ° C., a medium pH of 6.5-7.5, and a culture time of 10-16 hours are suitable.

The Bacillus amyloliquefaciens S13-3 strain cultured as described above can be used without being separated from the culture, and can also be used after separating the cells by a centrifugation method. Further, when this is used as a plant control agent, various additives such as a surfactant (for example, polyoxyethylene sorbitan monolaurate; Tween 20) and a spreading agent (for example, Approach BI manufactured by Maruwa Biochemical Co., Ltd.). They can be formulated together and used as granules, emulsions, wettable powders, flowables and the like.

By applying the bacterial cell or culture of the Bacillus amyloliquefaciens S13-3 strain thus prepared to a plant body or soil, it is possible to control plant diseases. The control agent of the present invention has a remarkable effect in controlling grape diseases caused by, for example, grape gray mold fungus, grape late rot fungus, grape white coat fungus, and the like, and plants other than grapes such as strawberry and cucumber Also effective in controlling gray mold and anthracnose fungi that infect mosquitoes.

The application method of the control material is appropriately selected depending on the form of the preparation, the type of the host plant, the type and degree of the disease, for example, a method of spraying the liquefied control agent on the ground or in the air, or directly spraying on the leaves of the plant, There are a method of coating, a method of dipping in a control solution, and the like.

The application rate varies depending on the type of disease and the applied plant, but is, for example, 5 L per field 1a at a concentration of 1.0 × 10 ⁸ cells / ml (0.02% Tween 20).

EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, of course, this invention is not limited to these Examples.

[Example 1: Culture example]
A single colony of Bacillus amyloliquefaciens S13-3 strain according to the present invention is platinum-inoculated in SCD medium (soybean casein digest broth), and cultured by shaking at 37 ° C, 150 rpm for 12 hours to obtain a culture solution It was. This culture solution was adjusted with SCD medium so as to contain 1.0 × 10 ⁸ cells / ml of S13-3 strain, and Tween20 (final concentration 0.02%) was added as a surfactant to obtain a flowable agent.

[Example 2: Small-scale spraying test for late grape rot]
In this example, an evaluation test for the control effect of Bacillus amyloliquefaciens S13-3 strain against grape late rot fungus was performed. A flowable preparation prepared in Example 1 above, using the planted Semillon grapes (shelf-made) at the Breeding Experiment Field (Tsukahara, Kofu City, Yamanashi Prefecture) attached to the University of Yamanashi Wine Science Research Center. We investigated by spraying. In this test field, various European brewing grapes are grown. Among them, Semillon variety is very vulnerable to grape late rot. The semillon seeds were grown in pesticide-free cultivation, and the control effect evaluation test of the S13-3 strain was conducted using the severity of grape late rot occurring naturally as an index. From July 7, 2010 to September 8, 2010, the flowable preparation prepared in Example 1 above is directly sprayed onto the grape bunches once a week every week, and the spray amount is 10 ml. / A bunch. (About 5L per 1a). As a control plot, an untreated plot where nothing was sprayed was set. After the completion of the spraying test, the disease severity (%) was calculated as <late rot diseased grains / whole grain × 100>.

On September 15, 2010, an investigation of the morbidity of late rot was performed, and based on the number of grains in which late rot appeared, the incidence was calculated by entering into the above formula.

From the results of Table 2 above, it was revealed that the Bacillus amyloliquefaciens S13-3 strain has a high control effect against grape late rot fungi.

[Example 3: Small scale spray test for strawberry anthracnose]
In this example, an evaluation test for controlling Bacillus amyloliquefaciens S13-3 strain against strawberry anthracnose was performed. In IAI Test Field Co., Ltd. (Oba, Shimizu, Shizuoka City, Shizuoka Prefecture), the strawberry (variety: Akihime) growing in the pot is used as a prototype, and the fungus prepared in Example 1 above is displayed. A dressing approach BI (manufactured by Maruwa Biochemical Co., Ltd.) was added at a concentration of 0.2% and sprayed as a flowable agent.
The whole strawberry seedling was sprayed once a week for 3 weeks from August 24, 2010 to September 7, 2010. At the same time, chemical control pesticide anthracol granule wettable powder (manufactured by Bayer Crop Science Co., Ltd.), commercially available microbial pesticide Biotrust wettable powder (manufactured by Idemitsu Kosan Co., Ltd.), Bacillus subtilis KS1 strain (patent document 5) ) 1.3-1.5 × 10 ⁸ cells / ml, and 5 untreated plots in which only water was sprayed without chemicals were prepared. In addition, each test group placed 25 strains of strawberry seedlings with an anthrax-carrying strain at the center of each strain, and the test was carried out in two iterations. Moreover, Approach BI 0.2% was applied to these materials as a spreading agent.

On September 28, 2010, we conducted a survey of strawberry anthracnose disease.
(a) After the start of spraying the drug, investigate the presence or absence and degree of chemical damage and dirt as appropriate.
(b) Surveyed strains: 15 strains were randomly selected from the strains not affected by the adjacent plots, and the surveyed leaves were observed in almost the same leaf position and stems in each plot as much as possible, and the number of healthy leaves was confirmed. .
(c) Survey item: The number of diseased leaflets (rate) was investigated, or the number of lesions per leaflet was investigated.
Disease index 0: No lesion 1: Small spot on leaf 2: Large lesion on leaf 3: Dwarf 4: Death
A five-step evaluation was performed, and the severity of the disease was calculated by (Equation 1).
Disease severity = (Σ (attack index × number of strains by disease)) / (4 × number of strains) × 100 (Equation 1)

Investigation results of anthracnose disease on strawberry
^* 1: Average of all surveyed strains (2 replicates: 30)

From Table 3 above, the incidence of S13-3 strain is low compared to biopest, which is a microbial material similar to the chemical pesticides Anthracol and Bacillus amyloliquefaciens S13-3. The result was highly effective. Since there were few lesions of anthracnose on stems and leaves and there was no phytotoxicity with spreading agents, it became clear that S13-3 strain was very useful as a plant disease control material.

[Example 4: Strawberry cut leaf spray test]
S13-3 strain cultured in SCD medium is 1.0 × 10 ^{8 in} SCD medium.
Adjusted to cells / ml. Leaves were cut from a commercially available strawberry seedling (variety: Nyoho) and sprayed with a Bacillus amyloliquefaciens S13-3 strain at a concentration of 2 ml / plate. At the same time, chemical pesticide getter wettable powder (active ingredient: Dietofencarb, manufactured by Nippon Soda Co., Ltd.), commercially available microbial pesticide Tough Pearl (produced by Idemitsu Kosan Co., Ltd.) The test was conducted in 4 test areas. The spraying concentration and frequency were performed according to the instructions of each manufacturer.
After natural drying, a small hole is made with a needle in the center of the cut leaf, and the flora disc of the strawberry anthracnose fungus Colletotrichum gloeosporioides grown on PDA medium (agar containing potato extract and glucose) is drilled. It was allowed to stand on the top so that the flora was on the bottom. After lightly moistening the cut leaves with sterilized water, the cells were cultured for 6 days under light irradiation conditions at 28 ° C. for 24 hours.
As the disease incidence index, 0: no lesion, 1: disease group just under the inoculated flora, 2: disease group twice the flora, 3: disease group three times the flora, 4: flora Eight grades were evaluated: 4 times disease group, 5: disease group 5 times the fungus flora, 6: disease group 6 times the flora, 7: disease group 7 times the flora.

The results are shown in FIGS. The Bacillus amyloliquefaciens S13-3 of the present invention showed the same level of control effect as that of the commercially available microbial pesticide Tafpearl adapted to strawberry anthracnose.

[Example 5: Evaluation test of antibacterial components of Bacillus amyloliquefaciens S13-3 strain]
In this example, a control effect test for Bacillus amyloliquefaciens S13-3 strain against grape late rot fungus was performed. First, the pathogenic bacteria to be assayed were cultured on PDA medium at 25 ° C. for 4 days. Next, the grown mycelium was punched out with a cork borer into a 4 mm diameter disc and placed in the center of a new petri dish containing PDA medium. Furthermore, Bacillus amyloliquefaciens strain S13-3 was linearly applied to the end of the same petri dish, and the culture of the test bacteria and Bacillus amyloliquefaciens strain S13-3 were carried out at 25 ° C. for 4 days. The formation of the inhibition band was observed.

FIGS. 5 to 7 are photomicrographs (200 ×) of the mycelial tip after culturing the test strain C. gloeosporioides at 25 ° C. for 3 days. This photograph shows that the hyphae of grape late rot fungus swells and bursts, and the growth of the pathogenic fungus is suppressed. It was considered that the antibacterial substance produced by the Bacillus amyloliquefaciens S13-3 strain of the present invention was involved in this growth inhibition. Then, when the antibacterial substance produced by the S13-3 strain was analyzed by high performance liquid chromatography (HPLC), it was identified as Iturin A, which is known as an antifungal agent. The amount of iturin A secreted into the medium when cultured in SCD medium for 6 days was 0.1 mg / ml, which was sufficient to suppress the growth of C. gloeosporioides. Therefore, it was clarified that the Bacillus amyloliquefaciens S13-3 strain suppresses late-rot fungi by producing and secreting iturin A outside the body.

[Example 6: Applied pathogen test]
In this example, a test for controlling effect of Bacillus amyloliquefaciens strain S13-3 against various fungal phytopathogenic fungi was performed. According to the method of Example 5, the calibration of the test bacteria was carried out by carrying out counterculture with the S13-3 strain in a PDA medium.

As a result of counter-culture between Bacillus amyloliquefaciens strain S13-3 and each pathogen, Alternaria brassicicola, Aspergillus niger, Botrytis alli, Botrytis cinerea, Botrytis fabae, Cochliobolus sativus, Cochliobolus miyabeanus, Colletotrichum coccodesgen, thulium Colletotrichum higginoianum, Helicobasidium mompa, Mucor racemosus, Mycosphaerella pinodes, Phytophthora porri, Pyricularia zingiberis, Pythium aphanidermatum, Rosellinia necatrix, Stemphylium lycopersici, Taphrina mume, Thielaviopsis basic It was. These pathogens are not only fruit trees such as grapes, apples and pears, but also fruit vegetables such as strawberries, cucumbers and other cucurbitaceae, and leafy crucifers. From this, it is clear that the S13-3 strain is a very useful antagonist.

[Example 7: Drug resistance test]
In this example, a resistance test against chemical pesticides of Bacillus amyloliquefaciens S13-3 strain was performed. First, an SCD medium containing the target chemical pesticide is made in a petri dish. The chemical pesticide concentration (ppm) at this time is the same as the general spray concentration. Next, Bacillus amyloliquefaciens strain S13-3 was applied to the surface of the medium containing the chemical pesticide so as to grow 50 to 100 colonies per plate and cultured at 37 ° C. for 1 day. As a control, the same amount of Bacillus amyloliquefaciens S13-3 was applied to an SCD medium containing no chemical pesticide. As a result of comparing the number of colonies grown after culture with the control group, it was revealed that the Bacillus amyloliquefaciens S13-3 strain was resistant to the following chemical pesticides.
200 ppm azoxystrobin, 200 ppm benomyl, 100 ppm dietofencarb, 100 ppm phenhexamide, 400 ppm fenitrothion, 1,000 ppm focetyl, 200 ppm cresoxime methyl, 300 ppm mandipropamide, 100 ppm procimidone, 200 ppm tricloprid, 200 ppm thiophanate methyl, 500 ppm trifloxystrobin, but 400 ppm carbaryl It is added that the appearance rate of colonies was significantly low for 100 ppm iprodione, 100 ppm maneb, and 700 ppm manzeb.
From the above results, it was shown that Bacillus amyloliquefaciens strain S13-3 is widely resistant to chemical pesticides commonly used in vegetables and fruit trees.

The Bacillus amyloliquefaciens strain S13-3 according to the present invention is effective for diseases of a wide variety of crops such as white rot, gray mold, late rot, and anthrax. This wide range of control effects can reduce the types and application rates of chemical and microbial pesticides that are currently applied to control each crop disease. The contribution to the realization of sustainable agriculture is very large. Therefore, industrial use as a microbial material is possible.

NITE P-1121

Claims (3)

Bacillus amyloliquefaciens S13-3 (NITE P-1121), a novel microorganism belonging to Bacillus amyloliquefaciens. A plant control material comprising a culture of Bacillus amyloliquefaciens strain S13-3 and / or a microbial cell as an active ingredient. The plant disease control material according to claim 2, wherein the culture and / or microbial cell of the Bacillus amyloliquefaciens S13-3 strain acts on grape late rot, gray mold, and strawberry anthracnose.