JP2827094B2 - Pest control method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to the use of a phenol-resistant fluorescent bacteria which does not produce an antibacterial substance, inoculation of the bacteria into seeds, and cultivation of seedlings in a soil containing at least 10 ⁵ cfu / g of the bacteria. The present invention relates to a method for controlling bacterial wilt, which aims to improve agricultural productivity under environmental protection.

[0002]

2. Description of the Related Art In recent years, the importance of ecologically friendly agriculture has been emphasized in the field of agriculture as the tendency toward environmental conservation on a global scale has increased. The establishment of a pest control technology harmonized with ecosystems that do not use pesticides has become an important issue. Under these circumstances, the national and public administrations have decided to use “organic agriculture”, “ecosystem-based agriculture”, and “sustainable agriculture”.
Measures that emphasize ecosystem safety, such as promotion of
Research and businesses in this area are increasing. Against this background, great expectations are placed on pest control by microorganisms.

[0003] Many microorganisms which are currently used for research on pest control or partially used are microorganisms which are normally present in nature.
It has been empirically known that such microorganisms existing in nature are generally less harmful to humans and fish and shellfish. In addition, microorganisms existing in nature are considered to act specifically on the target harmful microorganisms because the range of action is limited, and it is possible to minimize disruption to ecosystems. Microorganisms that have been studied and are partly commercialized have common properties. That is, microorganisms capable of producing substances that inhibit the growth of pests such as antibacterial substances or selective toxic substances are selected and used. In order to facilitate the selection or cultivation of microorganisms with enhanced capabilities, antibiotic-resistant bacteria are created, and furthermore, the pest control effect is achieved by making full use of techniques such as transfer of antimicrobial substance-producing plasmids. There is also a study on ways to further increase the quality. However, when these microorganisms are actually used in the field, the pest control effect, that is, the effect stability and the sustainability are remarkably small as compared with the pesticides and the like, and the control effect is often not exhibited at all depending on the plant growth environment.

[0004]

The problem to be solved by the present invention is to use a microorganism which survives in the natural world, which has little harm to humans and fish and shellfish, and which is used as a representative of the bacterial wilt disease of crop soil bacteria. It is to stably and continuously control the disease. The present inventors have been conducting long-term research on the control of bacterial wilt using microorganisms. It was concluded that it was difficult to ensure the stability and sustainability of the effect in microbial searches using substance production as an index. In other words, among microorganisms searched from general soil, rhizosphere soil, root surface, etc., there are some microorganisms that show a remarkable disease-inhibiting effect in vitro, but the effect is expressed even temporarily in actual fields. That was rare. This is because the manifestation of the effect is greatly affected by the environment in which the microorganisms are placed, that is, the physical and chemical properties of the soil, fertilizer management, water management, etc. As a result, the stability and sustainability of the effect manifestation Cannot be maintained. Therefore, the present inventors have focused on bacteria that inhabit the plant roots, which are relatively little affected by such external factors.

In general, it is well known that plants accumulate phenolic substances in living organisms due to external stress. For example, β-glucosidase activity in a living body of a plant is increased by invasion of a microorganism, and phenols are released from a phenol glycoside in the living body. These phenols are converted into quinone compounds by polyphenol oxidase or the like, and exhibit an inhibitory effect on bacteria. Therefore, when a microorganism invades a plant organism, the microorganism is exposed to a phenol compound or a quinone compound in a larger amount than usual. Therefore, it was considered that microorganisms need to have resistance to phenols in order to survive in plants.

[0006]

As a result of searching for bacteria based on such thinking, it was found that fluorescent bacteria having phenol resistance and not producing antibacterial substances are effective in controlling bacterial wilt. The present invention has been completed based on such knowledge. That is, the present invention may have a phenol resistant, and anti <br/> bacteriostatic substances non-producing fluorescent bacteria (fluorescent bacteria King A Agar
Medium, King B agar medium, ISP-II agar medium and
After inoculating on a tetextrose agar medium, plate culture is performed.
Proliferate more. On the other hand, Staphylococcus aureus (indicator)
On a Waxman agar medium, the bacterial wilt (indicator bacterium)
Pour on Le Mans agar to prepare plate culture medium. These two
Fluorescent cells produced in the above four types of media on a plate medium.
Bacterial culture (fluorescence punched with a 10 mm inner diameter bowler)
Agar medium containing bacterial colonies) were placed separately on each
If the culture is performed for one week,
The growth-inhibiting circle of the indicator bacteria is formed around the fluorescent bacterial culture.
No growth and wither on potato dextrose agar
Bacteria and lawn leaf rot fungi (indicating bacteria) and punching with the borer
The four fluorescent bacterial cultures were confronted at 28 ° C for 2 hours.
If weekly culture is performed,
A bacterium that does not form a growth inhibition zone against the indicator bacterium even on the ground;
Abbreviated below) . The present invention further have a phenol resistant,且
The present invention relates to a method for controlling bacterial wilt, which comprises inoculating seeds with fluorescent bacteria that do not produce antibacterial substances. If Furthermore, the present invention, have a phenol resistant, and relates to bacterial wilt control method characterized by seedling with soil to the antibacterial substances non-producing fluorescent bacteria comprising 10 5 ^cfu / g or more.

[0007]

The method for controlling bacterial wilt of the present invention will be described below in more detail. In the present invention, the phenol-resistant fluorescent bacteria are a monophenol structure hydroxybenzene 1,000 mg / l and a diphenol structure on an agar plate inoculated on a potato dextrose agar medium.
Filter paper disc for antibacterial substance assay impregnated with 0.1 ml of a mixed solution of 1,000 mg / l of 1,3-benzenediol and 1,000 mg / l of 1,2,3-benzenetriol triphenol structure (8 mm outer diameter: Toyo (Made of filter paper), and after culturing at 25 ° C. for 3 days, refers to bacteria growing without forming a growth inhibition circle around the disk.

[0008] The fluorescent bacteria which do not produce antibacterial substances are
The following bacteria are referred to. That is, the fluorescent bacteria are inoculated on King A agar medium, King B agar medium, ISP-II agar medium and potato dextrose agar medium, and then grown by plate culture. Separately, Staphylococcus aureus (indicator) is mixed with a Waxman agar medium, and bacterial wilt (indicator) is mixed with a Kerman agar medium to prepare a plate culture medium. Fluorescent bacterial cultures (agar medium containing fluorescent bacterial colonies punched with a 10 mm inner diameter bowl) produced in the above four types of culture media were separately placed on the two plate culture media, and cultured at 28 ° C. for one week. When performed, do not form a growth inhibition circle of the indicator bacteria around any fluorescent bacterial culture during the culture period, and, on a potato dextrose agar medium, wilt fungus and turf leaf rot fungus (indicating bacteria) and the above When two types of fluorescent bacterial cultures punched out with a borer are opposed to each other and cultured at 28 ° C. for 2 weeks, it refers to a bacterium which does not form a growth inhibition zone against the indicator bacterium on any medium during the culturing period.

The fluorescent bacteria used in the present invention are generally present in a large amount in plants, especially in roots, and can be easily isolated by the following method. That is, the fluorescent bacteria of the present invention grow in the roots of vegetables such as tomato, eggplant, cucumber, and pepper. Therefore, as a method for searching for fluorescent bacteria of the present invention, first, the roots of these vegetables are thoroughly washed with water, homogenized after surface sterilization, and the resulting triturated solution is potato dextrose agar medium, King B agar medium, and then plate culture. I do. After the colony is formed, the culture is irradiated with ultraviolet light, and fluorescent bacteria are detected by the presence or absence of fluorescence around the colony or around the colony. Next, the fluorescent bacteria may be detected and separated according to the method defined above.

[0010] The fluorescent bacteria of the present invention thus isolated exert an excellent effect on bacterial wilt in an actual field. The difference between many fluorescent bacteria studied so far and the fluorescent bacteria of the present invention lies in the ability to produce antibacterial substances,
The fluorescent bacteria of the present invention did not show any antibacterial activity as defined in the present invention against bacteria or filamentous fungi in a microorganism growth medium used for ordinary substance production and the like. Therefore, the antibacterial activity of the fluorescent bacteria of the present invention against bacterial wilt was not observed in vitro. However, as a result of raising seedlings of the plants inoculated with the fluorescent bacteria of the present invention and then planting them on soils infected with bacterial wilt and carrying out a disease inhibition test, a remarkable disease-inhibiting effect was observed and the effect was maintained.

Next, an embodiment of the present invention will be described.
The fluorescent bacterium of the present invention may be cultured according to a conventional method.If a large-scale cultivation is desired, the fluorescent bacterium of the present invention isolated as described above may be cultured in a large amount by the usual liquid culture method. Can be propagated. In a further preferred embodiment of the present invention, the plant cells in which the fluorescent bacteria survived during the immobilization, co-existing, immobilized using a polymer such as sodium alginate, polyacrylamide, carrageenan,
This is to perform immobilized culture. For example, when the fluorescent bacteria have settled in the rice root, the root culture root or callus of rice obtained by a conventional method may be mixed well with the fluorescent bacteria of the present invention, and then immobilized after homogenization.
As the fluorescent bacteria of the present invention cultured in this manner, a culture solution containing the cells can be used as it is, or only the cultured cells can be collected and used. It may be used in liquid form or may be used after drying. In still another embodiment, a liquid or dried product containing bacterial cells can be used in combination with a soil cultivation material, a rock wool material, a soil improving material such as silica, zeolite, vermiculite, diatomaceous earth, and sand. The cells of the present invention can be used as a mixture with other carriers. When such a cell of the present invention is used in combination with other carriers such as a soil conditioner, it is desirable to contain 10 ⁵ cfu / g or more cells from the viewpoint of maintaining cell stability or maintaining the effect. .

In the method of the present invention, the above-described fluorescent bacteria of the present invention may be sprayed and mixed on the soil before planting, or may be sprayed after planting. The effect of the present invention is best. One of the methods for exerting the effect is to inoculate seeds with the fluorescent bacteria of the present invention. The inoculation method is a method of sufficiently immersing an appropriate amount of seed in a solution containing the fluorescent bacteria of the present invention. It is particularly preferred to do this under reduced pressure. More specifically, 1 ml to 10 ml of a seed is mixed and immersed in 100 ml of the solution containing the fluorescent bacteria of the present invention at 10 ⁷ cells / ml or more. An immersion time of at least 1 hour is sufficient. According to such a method, the fluorescent bacteria of the present invention are inoculated into seeds and exhibit an excellent effect on bacterial wilt.

A further advantageous method of the present invention is a method in which the fluorescent bacteria-containing substance of the present invention is added to and mixed with a culture medium, and a seedling is grown on the culture medium. It is necessary that the concentration of the fluorescent bacteria of the present invention in the cultivation soil is 10 ⁵ cfu / g or more. When the bacterial concentration is low, the bacterial wilt cannot be sufficiently controlled.
When seedlings are planted after raising seedlings in such a soil, the incidence of bacterial wilt significantly decreases. Further, when the above-mentioned soil is sprayed or added and mixed, the fluorescent bacteria of the present invention is preferably 10 ⁴ cfu or more per 1 g of rhizosphere soil from the viewpoint of sustaining the effect. Furthermore, it is most effective to use the above-mentioned seed inoculation method and the method of soil cultivation in combination in a field where bacterial wilt frequently occurs to plant seedlings that have been raised.

[0014]

The present invention will be described in more detail with reference to the following examples. (Example 1) Plants of tomato (A), eggplant (B), cucumber (C) and sansho (D) cultivated from an institutional cultivation field in Hyogo prefecture, and botanical gardening (E) and green onion (F) of open-field cultivation The body was collected. The collected plant body is cut into about 5 cm after washing the roots with water,
About 10 g of the cut roots were washed by stirring with a screw mixer using 50 ml of sterilized water and 0.005% aerosol OT (manufactured by American Cyanamit Co., Ltd.). Stir and wash the roots in an 80% ethanol solution for 1 minute, then in a 1% aqueous sodium hypochlorite solution for 10 minutes, sterilize the root surface, wash with sterile water, and isolate the root microbial isolation source. did. 1g
After further cutting to about 5 mm, put in 20 ml of sterilized water, 10,000 rp
After homogenization for 15 minutes at 1 m, 1 ml of the triturated solution was diluted with 9 ml of potato dextrose agar medium and poured into a Petri dish. The same operation was performed on King B agar medium. Culturing was carried out at 15 ° C to 40 ° C depending on the soil temperature at the time of plant collection. 15 ℃
After culturing at 40 ° C. for 5 days, both cultures were irradiated with ultraviolet rays of 365 nm to identify colonies or colonies that produced a fluorescent substance in the surrounding area.

Next, the resistance of the separated fluorescent bacteria to phenolic compounds was tested using hydroxybenzene, 1,3-benzenediol and 1,2,3-benzenetriol. 1 ml of the cell suspension prepared to 10 ⁶ cells / ml of the separated fluorescent bacteria was diluted with 9 ml of potato dextrose agar medium and flattened, and then hydroxybenzene 1000 mg / l on an agar plate.
A filter paper disk (Toyo, trade name ADVANTEC, outer diameter 8 mm) impregnated with 0.1 ml of a mixed solution containing 1,000 mg / l of 1,3-benzenediol and 1000 mg / l of 1,2,3-benzenetriol was placed, and 25 After culturing at 3 ° C. for 3 days, the periphery of the disc was examined for the formation of growth inhibition circles.

Next, the antibacterial substance-producing ability of the strain which was found to be resistant to phenolic compounds was evaluated. First, King A agar medium, King B agar medium, ISP-II
Agar medium, potato dextrose agar medium
To 9 ml, 1 ml of the cell suspension of the above phenol-resistant strain prepared at 10 ⁶ cells / ml was mixed, flattened, cultured at 25 ° C, and after colony formation, further cultured for 48 hours. Was.
After the culture, the agar portion including the colony was punched out with a borer having an inner diameter of 10 mm. Thereafter, this was used as an antibacterial substance assay disk. The presence or absence of antibacterial substances against the indicator bacteria Staphylococcus aureus and the bacterial wilt fungus was determined to be 10 ⁸ cells / m
1 ml of the cell suspension of the indicator bacterium prepared in 1 l was mixed with 9 ml of Waxman agar medium for Staphylococcus aureus, and 9 ml of Kerman agar medium for Bacterial wilt and Petri dishes. (90 × 20 mm) and solidified. At the same time, each of the antibacterial substance assay disks was dispersed and placed at equal intervals in four places on a Petri dish (see FIG. 1).

The cells were cultured at 28 ° C. for 1 week, and the presence or absence of a growth-inhibiting circle against the indicator bacterium around the test disk for antibacterial substances was tested. The presence or absence of antibacterial substances against the indicator wilt fungus and lawn leaf rot fungus was determined by pouring a potato dextrose agar medium into a Petri dish, solidifying the wilt fungus grown on the filter paper disc in the center of the Petri dish. Alternatively, a lawn leaf rot fungus was placed, and the antibacterial substance assay disk was placed on the four sides at equal distances (see FIG. 2).
After culturing at 28 ° C for 2 weeks, the presence or absence of growth inhibition zone formation for the indicator bacteria was examined. Table 1 shows the presence or absence of the production of antibacterial substances by fluorescent bacteria having phenol resistance.

[0018]

[Table 1] Note) (Kinku A medium), (Kinku B medium), (ISP-II medium), (Potato dextrose medium (PDA medium)) are shown as antibacterial substance production medium. + Indicates that a growth inhibition circle and a growth inhibition zone were formed. -Indicates that the growth inhibition circle and the growth inhibition zone were not formed.

Example 2 The fluorescent bacteria shown in Table 1 were tested for antibacterial activity against bacterial wilt. Pseudomonas 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. Fluorescent bacteria were grown at 25 ° C in potato dextrose slants.
The cells were cultured 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 the same Petri dish as in Example 1. After removing excess water from the Petri dish surface,
After inoculating a 1 cm streak and culturing at 30 ° C. for 48 hours, the presence or absence of a zone of inhibition formed around the test bacteria was examined.

Next, the bacterial wilt disease inhibition test of the test bacteria was carried out by examining three layers of an upper layer consisting of 5 ml of 0.8% agar, a middle layer consisting of 3 ml of sea sand, and a lower layer consisting of 15 ml of white agar medium excluding sucrose. This was performed in a culture bottle with a stopper of 15 cm in length. First, add 80 to this culture bottle.
Seeds (variety: Large Fukuju) sterilized with a 1% ethanol solution and a 1% sodium hypochlorite aqueous solution were sown. 4 at 28 ° C
After germination in a dark place for a day, 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 0.5 ml of a cell suspension prepared by controlling the bacterial wilt to 10 ⁸ cells / ml was inoculated. After inoculation, the temperature was changed to 35 ° C and cultivation was continued in an artificial weather vessel. The control value 4 weeks after inoculation of the bacterial wilt fungus was calculated. A section inoculated with only the bacterial wilt was provided as a control section. Each test group was performed in triplicate with 3 strains per group.

In the present invention, 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 Table 2 shows the in vitro antibacterial activity against bacterial wilt and the control value against bacterial wilt using sterile seedlings.

[0022]

[Table 2] Note) The degree of disease in the control plot is 65, and 0.0 indicates a plot with a higher disease level 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 3) Inhibition of disease-causing disease-causing soil using a material containing No. 40 as a phenol-resistant antibacterial substance-non-producing fluorescent bacterium of the present invention and No. 27 as a control bacterium The test was performed. 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 10 ⁸ cel of the present invention bacteria and control bacteria
The ls / ml cell suspension was mixed at 25 v / v% with respect to the heat-treated soil. After mixing, the mixture was allowed to stand for 2 weeks. As a control, non-inoculated heat treated soil (no treatment) was used. These soils were filled in a plug seedling tray (136 holes), and tomato seeds (variety: Momotaro) were sown. After watering, the seedlings were grown in a glass greenhouse (30 to 40 ° C) for 2 weeks. In addition, automatic watering was performed during the seedling raising period. After raising the seedlings, each plug seedling was planted in a container filled with bacterial wilt disease-causing soil in which bacterial wilt was isolated at 10 ⁷ cfu / g of soil (in the case of house Momotaro plug seedlings, all strains died two weeks after planting). did. The degree of disease after planting was investigated. The investigation method was the same as in Example 2. The number of test strains was 3 in 12 replicates in 1 section. The results are shown in Table 3.

[0025]

[Table 3]

(Example 4) A disease inhibition test was carried out on a bacterial wilt disease soil using each material. The variety of tomato seeds used was House Momotaro. A cell suspension of 10 ⁸ cells / ml was prepared using No. 69 of the fluorescent bacteria of the present invention shown in Table 2. 1 ml of tomato seeds sterilized with 1% sodium hypochlorite and 80% ethanol aqueous solution were immersed in 10 ml of the cell suspension, and immersion treatment was performed at room temperature for 4 hours. The cultivation soil used in Example 3 was placed in a plug seedling tray (136
(Holes), and 10 ml of sterilized water was irrigated per hole, and then the seeds subjected to the seeding treatment were sown. [Test zone]

Next, the cultivation soil was filled in a plug seedling tray, 10 ml of the above-mentioned cell suspension was added per well, and unsterilized tomato seeds were sown. [Test plot] In addition, the heat-treated soil used in Example 3
Cell suspensions of ⁷ cells / ml, 10 ⁶ cells / ml and 10 ⁵ cells / ml were individually mixed at 25 v / v%. After mixing, the mixture was allowed to stand at 25 ° C. for 2 weeks.
After standing, the number of bacteria of the present invention in each culture was measured using a King B agar medium and a potato dextrose agar medium containing 5 mg / l crystal violet. The material using the bacteria of the present invention contained 10 ⁶ cfu, 10 ⁵ cfu, and 10 ⁴ cfu of the bacteria of the present invention in 1 g of the soil according to the above concentration. This material was filled in a plug seedling tray, and non-sterilized tomato seeds were sown. [Test zone]

The culture medium containing 10 ⁶ cfu per gram of the fungus of the present invention was filled into a plug seedling tray, and seeded with a seeding treatment to inoculate sterilized tomato seeds. [Test Zone] In the test zone, 10 ml of sterilized water was irrigated per well. In all test plots, after sowing tomato seeds, temperature
The same amount of sterilized water was appropriately irrigated in an artificial weather vessel at 28 ° C. and 80% humidity, and seedlings were raised for 3 weeks. After raising the seedlings, they were planted in containers filled with the same bacterial wilt disease as in Example 3. Filling the cultivation soil in the tray for plug seedlings in addition to the test plot, irrigating 10 ml of sterilized water per hole, then sowing non-sterilized tomato seeds,
The seedlings were raised under the same conditions in an artificial weather vessel for 3 weeks. Its true leaves 4-5
Before planting the seedlings (untreated seedlings) in the same container filled with the bacterial wilt disease soil as in Example 3, the above cell suspension was put into the planting hole at 10 ⁵ cfu per 1 g of plug culture (planting volume). (Converted to the weight of the transplanted plug culture soil) .After planting in the container [test zone] and the container, the above cell suspension was applied to the container soil (depth 10 cm) to 10 ⁴ cfu per 1 g of container soil. A plot [test plot] was added so as to be as follows. In addition, a section in which the untreated seedlings were planted was provided as a control section. [Test plot] In each test plot, sterilized water was irrigated at the root of the plant after planting, and the test was started. Automatic irrigation was performed during the test period. All investigations were performed in the same manner as in Example 2. 1 test strain
The test was performed in three repetitions of six strains in each ward. The results are shown in Table 4.

[0029]

[Table 4]

Example 5 Using a white liquid medium,
Root culture of tomato (cultivar: House Momotaro) was performed at 25 ° C for 2 weeks by a conventional method. After the culture, the culture roots were transferred to 100 ml of a 5 times diluted white liquid medium, and one platinum loop of the fluorescent bacteria of the present invention No. 12 shown in Table 2 (isolated from tomato roots) was inoculated, and co-cultivation was performed. . After culturing for 14 days, a culture solution having a bacterial concentration of 10 ⁸ cells / ml was added at 25 v / v% to the heat-treated soil used in Example 3.
Inoculation was performed to prepare a culture medium containing 10 ⁷ cfu in 1 g of the fluorescent bacteria of the present invention. Then, the cultivated soil was filled in a plug seedling tray, seeded with unsterilized pepper seeds (variety: Green 300), and then raised in a glass greenhouse (30 to 40 ° C.) for 2 weeks. As a control, a white liquid medium diluted 5 times at a ratio of 25 v / v% was added to the heat-treated soil (no treatment), and then the pepper seeds were sown and grown under the same conditions as above. After the seedling raising, each plug seedling was planted in a field (Toyooka City, Hyogo Prefecture) where the occurrence of pepper wilt was observed. The disease incidence after planting was investigated. The number of test strains was 3 in 20 replicates per section. Table 5 shows the results.
In the present invention, the disease incidence was calculated by the following method. Disease incidence = (number of diseased strains / number of test strains) x 100

[0031]

[Table 5]

[0032]

The method for controlling bacterial wilt of the present invention comprises the use of phenol-resistant fluorescent bacteria which do not produce antibacterial substances, inoculation of seeds of the bacteria and culture medium containing at least 10 ⁵ cfu / g. A method for controlling bacterial wilt characterized by raising seedlings. By reducing the damage caused by bacterial wilt on agricultural products by such a method, it is possible to improve agricultural productivity under environmental conservation.

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram showing a method for testing the presence or absence of antibacterial substances produced against indicator bacteria, Bacterial wilt and Staphylococcus aureus, in Example 1 of the present invention.

FIG. 2 is a diagram showing a method for assaying the presence or absence of antibacterial substance production against wilt fungi and lawn leaf rot fungi as indicator bacteria in Example 1 of the present invention.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 Petri dish 2 Assay disk (agar medium disk on which the bacterium of the present invention is grown) 3 Filter paper disk (disk on which indicator bacterium is grown)

──────────────────────────────────────────────────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 (3)

(57) [Claims] [Claim 1] have a phenol resistant, and antibacterial substances non-producing fluorescent bacteria (fluorescent bacteria King A Agar, key
B agar medium, ISP-II agar medium and potato
After inoculating on a Straus agar medium, grow by plate culture
Let it. On the other hand, waxing Staphylococcus aureus (indicator)
Bacterial wilt (indicating bacteria) on Kerman's agar
The plate is mixed with a supernatant medium to prepare a plate culture medium. This plate culture
Fluorescent bacterial cultures produced on the four media on a substrate
(Fluorescent bacterial rollers punched with a 10 mm inner diameter bowler)
Agar medium containing a knee) separately and cultivated at 28 ° C for 1 week.
Cultivation, any fluorescent cells within the culture period
Around the bacterial culture does not form a growth inhibition circle of the indicator bacteria,
And wilt fungus on potato dextrose agar medium and
Lawn leaf rot fungus (indicating fungus) and 4 species punched with the bowler
Cultivation at 28 ° C for 2 weeks in confrontation with a class of fluorescent bacterial cultures
Performed on any medium during the culture period.
(A fungus that does not form a growth inhibition zone for the indicator bacterium) . 2. A have a phenol resistant, and antibacterial substances non-producing fluorescent bacteria (fluorescent bacteria King A Agar, key
B agar medium, ISP-II agar medium and potato
After inoculating on a Straus agar medium, grow by plate culture
Let it. On the other hand, waxing Staphylococcus aureus (indicator)
Bacterial wilt (indicating bacteria) on Kerman's agar
The plate is mixed with a supernatant medium to prepare a plate culture medium. This plate culture
Fluorescent bacterial cultures produced on the four media on a substrate
(Fluorescent bacterial rollers punched with a 10 mm inner diameter bowler)
Agar medium containing a knee) separately and cultivated at 28 ° C for 1 week.
Cultivation, any fluorescent cells within the culture period
Around the bacterial culture does not form a growth inhibition circle of the indicator bacteria,
And wilt fungus on potato dextrose agar medium and
Lawn leaf rot fungus (indicating fungus) and 4 species punched with the bowler
Cultivation at 28 ° C for 2 weeks in confrontation with a class of fluorescent bacterial cultures
Performed on any medium during the culture period.
2. The method for controlling bacterial wilt of claim 1, wherein the use of a fungus that does not form a growth inhibition zone for the indicator bacterium is to inoculate the seed with the fungus. 3. have a phenol resistant, and antibacterial substances non-producing fluorescent bacteria (fluorescent bacteria King A Agar, key
Ring B agar medium, ISP-II agar culture areas and Potetodeki
After inoculating on a Straus agar medium, grow by plate culture
Let it. On the other hand, waxing Staphylococcus aureus (indicator)
Bacterial wilt (indicating bacteria) on Kerman's agar
The plate is mixed with a supernatant medium to prepare a plate culture medium. This plate culture
Fluorescent bacterial cultures produced on the four media on a substrate
(Fluorescent bacterial rollers punched with a 10 mm inner diameter bowler)
Agar medium containing a knee) separately and cultivated at 28 ° C for 1 week.
Cultivation, any fluorescent cells within the culture period
Around the bacterial culture does not form a growth inhibition circle of the indicator bacteria,
And wilt fungus on potato dextrose agar medium and
Lawn leaf rot fungus (indicating fungus) and 4 species punched with the bowler
Cultivation at 28 ° C for 2 weeks in confrontation with a class of fluorescent bacterial cultures
Performed on any medium during the culture period.
2. The method for controlling bacterial wilt of claim 1, wherein the use of a fungus that does not form a growth inhibition zone for the indicator bacterium is to grow seedlings on a soil containing at least 10 ⁵ cfu / g of the fungus.