JP2827093B2 - Pest control material - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a bacterial wilt control material comprising a fluorescent bacterium which has the ability to produce crystalline 2,4-diacetylfluoroclucinol and has no resistance to antibiotics. The purpose of the present invention is to improve the productivity of agriculture under environmental protection by reducing the damage caused by bacterial wilt of crops.

[0002]

2. Description of the Related Art As a biological control method for soil microbial diseases, many studies have been made on fluorescent bacteria of the genus Aureus spp. Which produce antibacterial substances or cytophores. Recently, attention has been focused on research on biological control using Pseudomonas fluorescens (Pseudomonas putida) and Pseudomonas fluorescens. It has progressed to the stage. This shoot Demonas fluorescens has long been known for producing phenacidine-based antibacterial substances and the like. Also, in 1953,
Production of 4-diacetylfluorochlorocinol was observed, and the action as an antibiotic was tested. As a result of the assay, an effect as an antibiotic against quorum-positive bacteria including actinomycetes and filamentous fungi was recognized.

[0003] From these findings, in the 1980's, the use of shoot Pseudomonas fluorescens for the control of soil-borne fungal diseases in organisms was examined, and in the 1990's, in v.
Due to its antibacterial activity against fungal pathogens in itro, its use in biological control such as black rot, barley wilt, seedling wilt and wheat leaf wilt was proposed. On the other hand, for soil-borne bacterial diseases,
Although there are few reports, E. Levy et al. (Plant Pathology, 1992, 4
According to 1), it was reported that both viable cells and their metabolite 2,4-diacetylfluorochlorocinol have antibacterial activity against phytopathogenic bacteria and phytopathogenic fungi in vitro. In this report, E. Levy et al. Propose that Shoot Monas fluorescens can be used as a biological control method not only for fungal diseases but also for soil-borne bacterial diseases such as bacterial wilt and soft rot.
However, these are all based on the results of in vitro antibacterial tests, and have not yet reached a technology supporting practical application.

By the way, soil-borne diseases in vegetable cultivation in Japanese agriculture are divided into fungal diseases and bacterial diseases according to the degree of damage, and bacterial wilt is a representative of bacterial diseases. Until now, various measures have been taken with chemical pesticides and the like, but it is a disease that is difficult to deal with, and expectations for biological control are increasing from the viewpoint of environmental protection. But,
At present, it has not been put into practical use due to the delay in research on the mechanism of action.

[0005]

SUMMARY OF THE INVENTION Accordingly, the present inventors
As a result of various investigations to develop a material for controlling bacterial wilt which can be used as a material and has a reproducible bacterial wilt control effect in a real field, and has a long-lasting effect. The present invention has been completed. The present inventors have paid attention to biopesticides that have already been developed as microorganisms that can be practically used as biological control materials. Among them, the potential of Shoot 検 討 Monas fluorescens, which has been sold as a biopesticide for the purpose of controlling some fungal diseases, as a material for controlling bacterial wilt disease, was examined. May exhibit antibacterial properties against bacterial wilt
2,4-Diacetylfluorochlorocinol-producing bacteria were studied.

[0006] Other researchers have studied the genetic level of Shoot Pmonas fluorescens, which produces 2,4-diacetylfluorochlorocinol. In order to use Shoots Monas fluorescens as a biological control material, at least in v
It is necessary to search for a strain that stably produces 2,4-diacetylfluorochlorocinol in itro. In the report of E. Levy et al. Mentioned above, the biomolecule I of Shooto Monas fluorescens (according to the classification of Bergey et al.) <Bergey'smanual of systematic bacteriol
It was stated that as a result of creating a resistant selection bacterium with an antibiotic using ogy> as the parent strain, the selected bacterium produced a maximum of about 70 μg / ml of 2,4-diacetylfluorochlorocinol. The present inventors have conducted detailed studies on the bacteriological properties of 2,4-diacetylfluoroclucinol-producing bacteria retrieved from various sources and disease suppression tests using plants.

[0007] According to a series of studies, 2,4-diacetylfluorochlorocinol-producing bacteria isolated from soil, root surface and the like were subjected to solvent extraction from a culture such as a culture solution when examined according to the method of E. Levy et al. As a result, it was found that the strain was classified into a small-producing strain that had to confirm 2,4-diacetylfluoroculocinol and a bacterial strain producing crystalline 2,4-diacetylfluoroclucinol. The former was largely isolated from the root surface and the rhizosphere soil, and the production of 2,4-diacetylfluorochlorocinol was up to about 100 μg / ml, which was comparable to the resistant bacteria of E. Levy et al. The latter was mainly isolated from within the crop roots.

[0008] The fractionated 2,4-diacetylfluorochlorocinol is both in
In vitro, it exhibited antibacterial activity against quorum-positive and quorum-negative bacteria including actinomycetes and actinomycetes. However, a significant difference was observed in the disease-suppressing effects of the producing bacteria themselves. After raising seedlings of plants inoculated with both of them individually, the plants were planted on the bacterial blight disease-causing soil and a disease-inhibition test was carried out. Was also insignificant. However, the latter strain showed growth in the crop roots and a remarkable disease-control effect. Moreover, the former strain, even if it is a strain that produces relatively large amounts of 2,4-diacetylfluoroqulucinol, is greatly reduced in production capacity and growth capacity when preserved by subculturing, etc. This left a major problem in quality control.

[0009]

On the basis of the results of the study, a fluorescent bacteria having a crystalline 2,4-diacetylphloroglucinol-producing ability is subjected to an effect confirmation test in an actual field, and intensive studies are repeated. As a result, the present invention has been achieved. That is, the present invention relates to a fluorescent bacterium which has the ability to produce crystalline 2,4-diacetylphloroglucinol and does not exhibit resistance to antibiotics, ie, streptomycin.
100 μg / ml, nalidixic acid 50 μg / ml and
King B medium containing 50 μg / ml rifampicin
After inoculation of the strain, the cells are cultured at a temperature of 15 to 40 ° C. for 2 weeks.
The present invention relates to a bacterial wilt control material comprising fluorescent bacteria that cannot be grown even afterwards .

[0010]

The present invention will be described below in more detail. First, in the present invention, a fluorescent bacterium having the ability to produce crystalline 2,4-diacetylfluorculocinol is defined as 15 to 15 μm after inoculating the strain on a King B agar plate medium or a potato dextrose agar plate medium.
When cultured at a temperature of 35 ° C and irradiated with ultraviolet light (365 nm),
It refers to a bacterium that produces crystalline 2,4-diacetylfluorochlorocinol, which can be visually confirmed, within 4 weeks after culture. Bacteria that do not show resistance to antibiotics include streptomycin 100
μg / ml, nalidixic acid 50 μg / ml and rifampicin 50
After inoculation of the strain into King B medium containing μg / ml, 15-40
A bacterium that is cultured at a temperature of ° C. and does not grow even after 2 weeks. Such a strain of the present invention grows particularly in the roots of vegetables such as tomato, eggplant, cucumber, and pepper. Therefore, the strain of the present invention can be easily searched for by washing the roots of these vegetables well, homogenizing and culturing the roots, and irradiating the culture with ultraviolet rays.

[0011] The strain of the present invention searched in this way is fundamentally different from the strain created by E. Levy et al. That is, the strain created by E. Levy et al. Is streptomycin (2
(50 μg / ml), nalidixic acid (250 μg / ml) and rifampicic acid (75 μg / ml), whereas the strain of the present invention never grows in such a high concentration of antibiotic medium. The point is that proliferation is slightly observed at about 1/10 or less of this concentration. The fluorescent bacterium of the present invention, which has the ability to produce crystalline 2,4-diacetylfluorochlorocinol and does not show resistance to antibiotics, is capable of producing a crystalline 2,4-digestion even when this is subcultured. No extreme decrease in the production ability of diacetylfluorochlorocinol was observed, and it was confirmed that storage or cultivation was possible and that it could be used as a material for controlling bacterial wilt.

With respect to the material for controlling bacterial wilt of the present invention, a culture solution containing cultured cells obtained by subjecting the strains searched as described above to large-scale culture by liquid culture or solid culture may be used as the material. Alternatively, only the cultured cells may be collected and used as a material. These may be used in liquid form or may be used after drying. As still another embodiment, the cultivation of a cell-containing liquid or dried material, rock wool material, silica, zeolite,
It can be used in combination with a soil conditioner such as vermiculite, diatomaceous earth, and sand. As described above, when the cells of the present invention are used in combination with other carriers such as a soil conditioner, it is necessary to contain 10 ⁶ cfu / g or more of cells from the viewpoint of maintaining cell stability or maintaining the effect. It is.

The material for controlling bacterial wilt of the present invention is preferably prepared by replacing it with an inert gas such as nitrogen which has been deoxygenated after its preparation, and storing it tightly, or keeping it at a low temperature of 10 ° C. or less in order to prevent a decrease in the activity of the cells. It is desirable to save with. Regarding the method for applying the material for controlling bacterial wilt of the present invention, for example, when the material of the present invention is applied to seeds, a seeding method in which the seeds are immersed in a cell-containing liquid, a fine powder obtained by mixing the bacteria with a carrier and stirring the seeds A seed coat abrasion method of mixing, a soaking pressure reduction method of performing a soaking method under reduced pressure, or the like can be used. As a method of applying to the plug seedlings, the cell-containing solution or the fine powder, which is the material of the present invention, can be added to and mixed with the plug culture medium. Further, the material of the present invention may be added to and mixed with soil before planting the plant, or the material of the present invention may be sprayed after planting.

The bacterial concentration in the material for controlling bacterial wilt of the present invention requires at least 10 ⁶ cfu / g when used in a mixture with a carrier, and 10 ⁶ cells / ml for a liquid material. The above is desirable. The amount of the material of the present invention varies depending on the type of crop, the time of use, the form of the material, and the like.In the case of seed treatment, the bacterial cell suspension of the present invention 10 ⁸ cells / ml per 1 ml of seed is 10 ml. It is desirable to perform seeding treatment to a degree, and when added to and mixed with the plug seedling culture soil, the bacterial cells of the present invention per 10 g of culture soil 10 ⁶ cfu
It is desirable to add so that it becomes above from the point of effect. When added to the soil after planting, the bacterial cells of the present invention are preferably 10 ⁴ cfu or more per g of rhizosphere soil from the viewpoint of maintaining the effect.

[0015]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the invention is limited thereto. In the examples, all percentages are by weight unless otherwise specified.

(Example 1) Tomatoes (A), eggplants (B), cucumber (C), peppers cultivated from a facility cultivation field in Hyogo Prefecture
The root zone soil and the plant were collected from (D), the bok choy (E), the green onion (F), and the turf (G) at the golf course. The collected plant body is cut into about 5 cm after washing the roots with water,
Approximately 10 g of the cut roots are each 50 ml of sterilized water and 0.005% aerosol OT
(American Cyanamit Co., Ltd.) was used to perform stirring and washing with a screw mixer. The washing liquid was mixed to obtain a root microorganism separation source. The agitated and washed roots were immersed in an 80% ethanol solution for 1 minute, further immersed in a 1% aqueous sodium hypochlorite solution for 10 minutes to sterilize the root surface, washed with sterile water, and used as a root microbial isolation source. .

Separation operation was performed using the rhizosphere soil, the mixed washing solution, and the surface sterilized root as a microorganism separation source. The rhizosphere soil was shaken with 100 times the volume of sterile water for 10 minutes, and allowed to stand for 1 minute.
Dilute 00-fold and add 1 ml of the potato dextrose medium (agar
The mixture was pulverized with 9 ml (1.5%) and poured into a Petri dish (pour method).
Culturing was carried out at 15 ° C to 40 ° C depending on the soil temperature at the time of plant collection. 1 ml of the mixed washing solution was similarly cultured by the same pour method. 1 g of the surface-sterilized root was further cut into about 5 mm, placed in 20 ml of sterilized water, homogenized at 10,000 rpm for 15 minutes, and cultured with 1 ml of the ground solution by the same pour method. After 72 hours, each culture was replicated on each plate medium of King B agar medium and Pseudomonas selective separation P-1 agar medium, and cultured at 15 to 40 ° C. for 72 hours.
By irradiating with 5 nm ultraviolet rays, colonies producing a fluorescent dye were identified. The colony producing the fluorescent dye was again inoculated into King B liquid medium and potato dextrose liquid medium, and incubated at 25 ° C. for 3 weeks.

After completion of the culture, the culture is irradiated with ultraviolet light of 365 nm, and the culture in which the production of a yellow-green to yellow fluorescent substance is observed and the culture in which the production of crystals having blue-white fluorescence is observed are described below. Was performed. The culture solution in which the production of crystals was observed was separated by filtration with 5A filter paper (manufactured by Toyo). Other culture solutions were freeze-dried, and components soluble in a mixed solvent of chloroform-acetone (1: 1) were extracted from the dried product. The crystals and the extract were dried, suspended in a small amount of water and methanol, and the insolubles were separated by filtration, dried and weighed. The dried product was subjected to thin-layer chromatography (silica gel plate, benzene: chloroform: methanol =
4: 2: 1) The sample was subjected to analysis, and the dried product having spots with Rf values of 0.5 to 0.53 was measured by a nuclear magnetic resonance apparatus and a mass spectrometer. As a result, it was confirmed that the extract was mainly composed of 2,4-diacetylfluorochlorocinol, and the crystal was 2,4-diacetylfluorochlorocinol.

Next, the resistance to antibiotics of the strain in which the production of 2,4-diacetylfluorochlorocinol was recognized was examined using streptomycin, nalidixic acid and rifampicin. Streptomycin (100%)
μg / ml, nalidixic acid at 50 μg / ml, and rifampicin at 50 μg / ml were mixed in a King B agar medium and poured into a Petri dish. After removing excess water on the agar surface, the produced bacteria were streaked, cultured at 25 ° C for 3 weeks, and examined for growth. Table 1 shows the production amount and the triple resistance of the 2,4-diacetylfluorochlorocinol-producing bacteria.

[0020]

[Table 1]

Example 2 Antibacterial activity against bacterial wilt was tested on 2,4-diacetylfluorochlorocinol-producing bacteria shown in Table 1. Shoots as tomato bacterial wilt fungus: Monas solanacearum (Pseudom
onas solanacearum, MAFF-03-01485) was cultured in a potato dextrose slant culture medium at 35 ° C. for 72 hours to obtain indicator bacteria.
2,4-Diacetylfluorochlorocinol-producing bacteria were cultured in a potato dextrose slant culture medium at 25 ° C. for 96 hours to obtain test bacteria. One indicator loop was suspended in 100 ml of sterile water, and 1 ml of the suspension was mixed with 9 ml of potato dextrose agar medium and poured into a petri dish. After removing excess water from the surface of the Petri dish, streak the test bacteria to about 1 cm in streaks and incubate at 30 ° C for 48 hours.The diameter of the inhibition circle formed at the periphery of the test bacteria (diameter perpendicular to the streaks) ) Was measured. As a result, all the test bacteria shown in Table 1 formed a growth inhibition circle against the bacterial wilt fungus, and 12 m in R-3, 7, 8, 9, and RS-16.
m or less, and the inhibition circle diameter of other test bacteria ranged from 35 mm to 49 mm.

Example 3 Among the 2,4-diacetylfluorochlorocinol-producing bacteria shown in Table 1, those strains excluding R-3,7,8,9, RS-16 were subjected to the same operation as in Example 2. Test bacteria were used. Inhibition test for bacterial wilt disease of the test bacteria. Culture bottle with outer diameter 2.5cm, height 15cm, consisting of three layers: upper layer: 5% 0.8% agar, middle layer: 3ml sea sand, lower layer: 15ml white agar medium excluding sucrose. I went in. First, in this culture bottle, tomato seeds sterilized with 80% ethanol solution and 1% sodium hypochlorite aqueous solution (variety: Large Fukuju)
Was sown. After germination at 28 ° C for 4 days in a dark place, the seeds were transferred to an artificial weather device and cultivated at 30 ° C for 3 days.

The culture bottle was inoculated with 0.5 ml of a cell suspension prepared at 10 ⁷ cells / ml of the test bacteria. After inoculation, cultivation was performed at 30 ° C. for 3 days, and then the bacterial wilt of Example 2 was cultured at 10 ⁸ cells / ml.
Was inoculated with 0.5 ml of the prepared cell suspension. 35 ℃ after inoculation
And continued cultivation in an artificial weather vessel. The disease control was measured 30 days after the inoculation of the bacterial wilt fungus, and the control value was calculated. As a control section, a section inoculated with only the bacterial wilt was provided. Each test group was performed in triplicate with 3 strains per group. The roots of the seedlings in each section were surface-sterilized by the same method as for seed sterilization, and the roots were cut to a length of about 2 cm and placed on a King B agar plate and a potato dextrose agar plate containing 5 mg / l of crystal violet. After culturing at 25 ° C for 7 days, King B agar plate was tested for the presence of the test bacteria by the production of fluorescent material around the colonies under 365 nm UV irradiation, and potato dextrose agar plate was fluorescent under UV irradiation. Habitats were assessed by crystal production. The root colonization state of the test bacteria was indicated by the root length ratio of the habitat to the total root length by the line crossing method as the root colonization degree. The results are shown in Table 2. still,
The degree of disease and the control value were calculated by the following methods. Disease degree = [(Σ disease index x number of strains) / (10 x total number of strains)] x 100 disease index: withering, 10; whole body withering, 5; partial withering, 3; yellowing, 2 control value = [(control group disease Degree-degree of disease inoculated with test bacteria) / degree of disease in control group] x 100

[0024]

[Table 2] Note) The degree of disease in the control plot was 93, and 0.0 indicates the plot with higher disease in the test plot than the control plot.

In the following examples, the cells of the present invention and the control cells used in the examples were cells cultured on a potato dextrose slant culture medium at 25 ° C. for 96 hours.

(Example 4) A material containing ER-32 as a crystalline 2,4-diacetylfluorochlorocinol-producing bacterium of the present invention in Table 2 of Example 3 and RS-19 as a control bacterium was prepared. A disease suppression test was carried out on a bacterial wilt disease soil. Commercial Rice nursery soil and horticultural soil 4: 1 mixture, after cooling to room temperature for 1 hour dry heat sterilized at 180 ° C., immediately present invention bacterium and 10 ⁸ c of the control fungus
Cell suspensions of ells / ml, 10 ⁷ cells / ml, and 10 ⁶ cells / ml were mixed at 25 v / v% to the heat-treated soil. After mixing, the mixture was allowed to stand at 25 ° C. for 2 weeks. After standing, the numbers of the bacteria of the present invention and the control bacteria in the soil were measured using King B agar medium and potato dextrose agar medium containing 5 mg / l crystal violet. The material using the bacterium of the present invention is 10 ⁷ cf / g.
u, 10 ⁶ cfu, and 10 ⁵ cfu of the present bacterium, and the material using the control bacterium contained 10 ⁸ cfu, 10 ⁷ cfu, and 10 ⁶ cfu of the control bacterium in 1 g. Also, sterile water was added to the cultivated soil (untreated 1) mixed with cultivated soil 4: 1 and further heat-treated cultivated soil.
Cultivated soil (no treatment 2) was added and mixed at a ratio of 5 v / v%. Filling these cultivated soil into plug seedling trays (136 holes), sowing tomato seeds (variety: Momotaro), crow greenhouse (30 ° C)
-40 ° C) for 2 weeks. In addition, automatic watering was performed during the seedling raising period. After raising the seedlings, each plug seedling was treated with bacterial wilt per gram of soil.
The plant was planted in a container filled with 10 ⁷ cfu-isolated bacterial wilt diseased soil (in the case of house seedlings of Momotaro, all strains died two weeks after planting). The degree of disease after planting was investigated. The investigation method was the same as in Example 3. The number of test strains is 12 in 1 ward
Three replicates of the strain were performed. The results are shown in Table 3.

[0027]

[Table 3]

Example 5 A cell suspension of 10 ⁸ cells / ml was prepared using ER-26 in Table 2 of Example 3. To 10 ml of this cell suspension, 1 ml of tomato seeds (variety: House Momotaro) sterilized with 1% sodium hypochlorite and an 80% aqueous ethanol solution was immersed, and immersed at room temperature for 4 hours. The heat-treated soil used in Example 4 was filled in a tray for plug seedlings, sufficiently irrigated with sterilized water, and then immersed in seeds. The treated seeds were sown and grown in an artificial weather device at 28 ° C for 3 weeks. A sterilized tomato seed was similarly raised as a control. After the completion of the seedling raising, the seedlings were planted in containers filled with the same bacterial wilt disease soil as in Example 4. The degree of disease after planting was investigated. The survey method is
Performed in the same manner as in Example 3. The number of test strains was 3 in 6 replicates per section. The results are shown in Table 4.

[0029]

[Table 4]

(Example 6) Table 3 of Example 3
The potato dextrose liquid medium diluted 5-fold was inoculated using ER-71 and allowed to stand at 28 ° C. for 2 weeks. After the culture, the cells were diluted 100-fold with sterile water. Tomato seedlings (cultivar: House Momotaro) which were plug-raised for 4 weeks in a 28 ° C artificial weathering vessel using commercially available seedling culture soil are planted with plugs in the same container filled with the bacterial wilt disease as in Example 4. The wells at the end were perfused with 50 ml of ER-71 diluted culture solution per well. As a control, there were provided a section (1) in which 50 ml of sterilized water was perfused and a section (2) in which 50 ml of a 5-fold diluted potato dextrose liquid medium was perfused.
After irrigation, tomato seedlings were planted and the degree of disease after planting was investigated. The investigation method was the same as in Example 3. The number of test strains was 3 in 6 replicates per section. Table 5 shows the results.

[0031]

[Table 5]

[0032]

EFFECT OF THE INVENTION The material for controlling bacterial wilt of the present invention comprises crystalline 2,4-
A bacterial wilt control material composed of fluorescent bacteria having a diacetylfluoroclucinol-producing ability and exhibiting no resistance to antibiotics. Use of such a material reduces damage caused by bacterial wilt of agricultural crops. By reducing, it is possible to improve the productivity of agriculture under environmental protection.

──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Yoshio Maekawa 3-491 Higashi-Jiyugaoka, Shizen-cho, Miki-shi, Hyogo (72) Inventor Taizo Akiyama 331-9 Shinzume, Yoneda-cho, Takasago-shi, Hyogo (72) Yoshinori Hayashi, Inventor 2-50 Kokukita, Inami-cho, Kako-gun, Hyogo Examiner Motohiro Okubo (58) Field surveyed (Int. Cl. ⁶ , DB name) A01N 63/00

Claims (1)

(57) [Claims] 1. A fluorescent bacterium having an ability to produce crystalline 2,4-diacetylphloroglucinol and showing no resistance to antibiotics, ie, 100 μg of streptomycin.
/ Ml, nalidixic acid 50 μg / ml and rifampi
The strain was placed in King B medium containing 50 μg / ml of syn.
After seeding, culture at a temperature of 15 to 40 ° C, and after 2 weeks
Bacterial wilt control material consisting of fluorescent bacteria that do not grow .