JP2002053414A - Inhibitor for soil disease and pest and method for inhibiting soil disease and pest - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an inhibitor for soil disease and pest capable of conveniently inhibiting soil disease and pest with high safety and without affecting adverse effects on the natural environment and provide a method for inhibiting soil disease and pest. SOLUTION: The inhibitor for soil disease and pest is obtained by including the cultured cell of a new microorganism, Gliocladium virens G2 (FERM P-17381) strain, or prepared by mixing the cultured cell of the strain with a bulking agent and a nutrient source. The method for inhibiting soil disease and pest is carried out by mixing these inhibitors with soil.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a soil pest control agent and a soil pest control method. More specifically, the present invention relates to a soil pest control agent and a soil pest control method which are simple, have high safety and do not adversely affect the natural environment.

[0002]

2. Description of the Related Art In agricultural production, the same crop is continuously cultivated on the same soil in order to maximize the yield on limited arable land. For this reason, the balance of the microflora living in the soil of the arable land is disrupted, and soil pests and insect pests are frequently occurring. Therefore, in order to control the soil pests, soil disinfection is performed before crops are planted.

[0003] In this soil disinfection, a gas agent which is highly toxic to the human body, such as methyl bromide or chlorpicrin, is usually used. Methyl bromide and chlorpicrin are used as soil disinfectants because these compounds have excellent bactericidal and insecticidal activity. Therefore,
Treating cultivated soil with these gas agents not only kills the pathogenic microorganisms that cause soil disease and insect pests, but also kills the useful microorganisms that inhabit the soil. in this way,
If the microorganisms in the cultivated soil are completely killed, there is a problem that after the effect of the soil disinfection has disappeared, the pathogenic microorganisms that have entered the soil are greatly proliferated. Methyl bromide is a substance that can diffuse into the atmosphere after use for disinfecting soil and cause destruction of the ozone layer. Law development is required.

[0004] In response to such demands, solar heat disinfection, hot water disinfection, and steam disinfection of soil are being studied. However, sufficient disinfection cannot be performed only by solar heat. There is a drawback in that it requires equipment for generating hot water and steam and consumes a large amount of energy. Therefore, as a method of disinfecting the soil without consuming a large amount of energy, a method of controlling soil pests that is safe and has a low environmental load by utilizing the functions of microorganisms is being studied. For example, in Japanese Patent Application Laid-Open No. 7-227293, a gliotoxin (Gliot) having an antagonistic action against soil pests is disclosed.
Proposal of a method for producing gliotoxin, comprising solid-culturing a bacterium of the species Gliocladium virens having the ability to produce oxin) and extracting gliotoxin from the solid culture. However, there is also a drawback that the productivity of gliotoxin varies greatly depending on the strain of the bacterium of the species Gliocladium virens.

[0005] Under these circumstances, there is a demand for the development of a safe and low environmental burden soil pesticide and a method for controlling soil pests by utilizing microorganisms capable of stably producing a large amount of gliotoxin. .

[0006]

SUMMARY OF THE INVENTION The present invention is simple, safe, and has no adverse effect on the natural environment.
An object of the present invention is to provide a soil pest control agent and a soil pest control method capable of controlling soil pests.

[0007]

Means for Solving the Problems The present inventors have conducted various searches in order to solve the above-mentioned problems, and as a result, have found that Gliocladium virions (Gliocladium virions).
ens) It has been found that the G2 [FERM P-17381] strain can produce gliotoxin in a large amount and stably, and the present invention has been completed based on these findings.

That is, the gist of the present invention is as follows. (1) Glioclium bilensu (Gliocl
adium virens) G2 [FERM P-17
381] A soil pest control agent containing a culture of a strain. (2) Glioclium bilensu (Gliocl
adium virens) G2 [FERM P-17
381] A soil pest control agent comprising a mixture of a culture of a strain, a bulking agent and a nutrient. (3) The soil pest control agent according to the above (2), wherein the nutrient source is one or more substances selected from the group consisting of glucose, ammonium sulfate, soybean flour, bran and flour. (4) The soil pest control agent according to any one of (1) to (3), wherein the soil pest is a soil pest caused by a filamentous fungus, a bacterium, a nematode, a nematode-borne virus, or a microorganism-borne virus. (5) Glioclium bilensu (Gliocl
adium virens) G2 [FERM P-17
381] A method for controlling soil pests, which comprises using a culture of a strain. (6) Glioclium bilensu (Gliocl
adium virens) G2 [FERM P-17
381] A method for controlling soil pests, which comprises using a mixture of a culture of a strain, a bulking agent and a nutrient. (7) The method for controlling pests and insect pests according to the above (6), wherein the nutrient source is one or two or more substances selected from the group consisting of glucose, ammonium sulfate, soybean flour, bran and flour. (8) The method for controlling soil pests according to any of (5) to (7), wherein the soil pests are soil pests caused by filamentous fungi, bacteria, nematodes, nematode-borne viruses or microorganism-borne viruses. (9) The method for controlling pests and insect pests according to any one of (5) to (8), wherein the soil is soil in a main field before planting or seedling culture before transplantation. (10) Gliocldium virens
adium virens) G2 [FERM P-17
381] strain.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The soil pest control agent of the present invention is Gliocladium bilens.
virens) G2 [FERM P-17381] strain [hereinafter abbreviated as Gliocladium virens G2 strain]. ] The soil pest control agent containing the culture of [1].

The morphological properties of the Gliocladium virens strain G2 have morphological properties unique to Gliocladium virens species. That is, the conidiophore has a colorless, upright, simple or branched form, and has a height of 60 to 150 μm. And, the primary branch metula is rotiferous or countersunk, its length is 6-15 μm, and on its most advanced phialide,
It forms a conidiospore mass. The conidiospores are pale green, the aggregate is dark green, have a broad elliptical or subspherical shape, are unicellular and slightly protrude at one end, and have a size of 2
５5 × 2 to 4 μm. In addition, chlamydospores are spherical or subspherical in shape and have a diameter of 7-12 μm.

[0011] The method for culturing the Gliocladium virens G2 strain can be performed by the same method as that for culturing a normal microorganism. Specifically, a liquid culture method using a reciprocating shaking culture, a jar fermenter culture, or the like, or a solid culture method can be used.
The medium components used in this culture are not particularly limited, and sugars such as glucose, sucrose and molasses, organic acids such as citric acid, alcohols such as glycerin as carbon sources, and ammonia, ammonium sulfate, and ammonium chloride as nitrogen sources. And ammonium salts such as ammonium nitrate and nitrates. In addition, as an organic nitrogen source, yeast extract, corn steep liquor, meat extract, wheat germ, polypeptone, soybean powder, and the like are used. Further, as inorganic salts, phosphoric acid, potassium chloride, calcium chloride, manganese sulfate, ferrous sulfate and the like can be used.

The culture conditions are preferably liquid culture or solid culture performed under aerobic conditions such as aeration stirring and shaking culture. The culture temperature is preferably in the range of 10 to 37 ° C.
5-32 ° C is more preferred. The culture period is 1 to
14 days, preferably 2 to 7 days. Furthermore, when culturing in large quantities, ordinary liquid culturing such as tank culturing may be used, or solid culturing using a porous material impregnated with plant-derived solid components such as bran, sugars and a nitrogen source, etc. May be adopted.

[0013] The culture thus obtained by culturing is:
Although the culture can be used as it is, the culture may be ground or shredded together with the medium. The cells may be used by scraping the cells from the medium in the culture, or the cells may be separated by centrifugation of the culture and used. further,
The pulverized product or cells of the culture collected here may be used as a dry powder by natural drying, spray drying, freeze drying, or the like. The dry powder preferably has a water content of 20% by mass or less, and preferably has a particle size of 5 mm or less.

The thus obtained culture of the Gliocladium virens strain G2, its crushed product or its dry powder can be used as it is as a soil disease and insect control agent. And nutrients are preferably used in the form of a mixture thereof.

The extenders used herein include kaolin clay, pyroferrite clay, bentonite, montmorillonite, diatomaceous earth, synthetic hydrous silicon oxide, acid clay, talc, clay, ceramic, quartz, sericite, vermiculite, pearlite, Otaniishi Mineral powder such as anthraite, limestone, coal ash, zeolite, and attapulgite are preferably used.

Nutrient sources include rice husk, bran, crab husk, shrimp husk, krill fine powder, rice bran, wheat flour, corn cob, peanut hull, bone meal, fish meal, meal powder, sawdust, wood powder, charcoal, kun Charcoal, bark charcoal, rice husk charcoal, plant ash, peat moss, grass charcoal, dried animal dung, activated carbon, oil cake, defatted soy flour,
Organic fine powder such as full fat soybean powder is preferably used.

Next, Gliocladium bilens G
When a mixture obtained by mixing a bulking agent and a nutrient source with the cultures of the two strains is used as a soil pest control agent, the mixing ratio of each of these components is determined by adding the bulking agent to 100 parts by mass of the dry powder of the culture. 0 to 95 parts by mass, preferably 5
The nutrient may be 1 to 99.9 parts by mass, preferably 5 to 99.5 parts by mass.

In addition, a liquid carrier can be used when a bulking agent or a nutrient is blended with a culture of the Gliocladium virens G2 strain to prepare a formulation. As the liquid carrier, water, vegetable oil, liquid animal oil, water-soluble polymer compounds such as polyvinyl alcohol, polyvinyl pyrrolidone, and polyacrylic acid are used. Further, if necessary, starch hydrolyzate and D-sorbitol, lactose, soluble extenders such as maltose, casein and gelatin, gum arabic, alginic acid, fixing agents and dispersants such as bentonite, propylene glycol and ethylene glycol Antifreezing agents, natural polysaccharides such as xanthan gum, and thickeners such as polyacrylic acid.

And this Gliocladium bilen
Culture of S. aureus G2 strain and soil containing the same as an active ingredient
As a pesticide, the concentration of bacteria is gliocladium.
1 × in the colony forming unit of the virens G2 cells
10^Two~ 1 × 10^Tencfu / g, preferably 1 × 10^Three
~ 1 × 10⁹cfu / g, more preferably 5 × 10 ^Three
~ 1 × 10⁸Cfu / g is preferably used.
You.

When the soil is treated with the soil pest control agent, the soil is preferably applied to the field soil before sowing or planting the seedlings, or to the seedling cultivation soil before sowing or transplanting the seedlings. In addition, the application rate of the soil pest control agent is 10 to 300 per 10 ares of the field soil.
kg.

The soil pests that can be controlled by treating the soil with the soil pest control agent include the fungi, bacteria, nematodes, nematode-borne viruses, and microorganism-borne viruses, which are used in the underground parts of plants. It works effectively for controlling the pests and insects caused by soil. Among these soil pests, for example, Cucurbitaceae, Solanaceae, Rosaceae, Leguminosae, Brassicaceae, Lily family, Asteraceae, Acalytheceae, Anthracnose, White silk disease, Root disease, Wilt, root wilt, chlorosis, vine wilt, acute wilt,
It is particularly effective in controlling soil diseases such as clubroot, seedling wilt, and wilt disease, and soil nematode damage caused by cyst nematodes, root knot nematodes, and nexare nematodes.

[0022]

[Examples 1 to 13]

(1) Selection of strains For various strains belonging to the genus Gliocladium, their gliotoxin productivity was evaluated, and the most useful strain was selected.

In this evaluation, ten strains shown in Table 1 were used. Each of these strains is inoculated with a seed strain cryopreserved in a potato dextrose medium, and
Shaking culture was performed at 8 ° C for 36 hours. The obtained culture solution was subjected to centrifugation at 3,000 rpm for 30 minutes using a centrifuge to remove precipitates, and further filtered through a 0.45 μm disk filter to remove the culture solution supernatant. Obtained.

Next, using 50 ml of the culture supernatant for each of these 10 strains, placing them in a separating funnel, adding 50 ml of chloroform, and collecting chloroform with stirring. By performing the reaction twice, a chloroform solution of gliotoxin was obtained. This gliotoxin chloroform solution was evaporated to dryness under reduced pressure, dissolved again in chloroform, and the gliotoxin content in the supernatant was measured by HPLC using an ODA column.

The results are shown in Table 1.

[0026]

[Table 1] From the results shown in Table 1, a Gliocladium virens G2 strain having a high ability to stably produce gliotoxin in the largest amount was selected.

(2) Production of a pest control agent for soil pests Inoculation of a seed strain of Gliocladium virens G2, which is cryopreserved in a potato dextrose medium,
Shaking culture was performed at 28 ° C. for 24 hours. 500 g of pressed wheat was used as a culture carrier, and 350 ml of a mixture of a culture solution and sterilized water was added to the pressed wheat.

Then, 36 cm in length, 22 cm in width, depth 6
On a tray of cm, push the oats with the culture solution and the thickness is about 5c.
m. The upper surface of this tray was covered with a lid having an air hole, and the plate was incubated at 28 ° C. for 10 days. During this culture period, the cells were cultured under light conditions, and the entire carrier was stirred on the third day from the start of the culture.

After completion of the culture, the culture was transferred to a drying tray, spread thinly to a thickness of about 1 cm, covered with filter paper, and dried with a dryer at 30 to 35 ° C. for 24 hours. This culture was dried and pulverized to obtain a bacterial dry powder.

Next, the dried powder of the bacterium was used in Table 2 by using dry peat as a bulking agent, and using wheat flour, bran, defatted soy flour, full fat soy flour, glucose, and ammonium sulfate as nutrient sources. By mixing at the indicated ratio, a soil pest control agent was produced.

(3) Evaluation of gliotoxin productivity of soil pest control agent As a model soil, sterilized Chinese vermiculite 5
To 0 g, 5 g of each of the soil pest control agents produced in the above (2) was mixed, and sterilized water was added to adjust the water content to 50% by mass, followed by filling in a biopot.

Next, this biopot was allowed to stand still in an incubator kept at 25 ° C.
0 ml of ion-exchanged water was added and stirred. Then, this was filtered, 50 ml of the filtrate was transferred to a separating funnel, and a gliotoxin extraction operation with 50 ml of chloroform was performed twice. The obtained extract was dried, dissolved in 0.7 ml of chloroform, and filtered through a 0.45 μm disc filter.
It was quantified by HPLC analysis. The analysis conditions here are O
Using a DS column, the flow rate was 1 ml / min, and the detection wavelength was 254 nm. From the analytical values thus obtained, the amount of gliotoxin produced per 100 g of model soil was calculated. The results are shown in Table 2.

[0033]

[Table 2]

(4) Test for Controlling Rhizoctonia Seedling Damage As a soil disease, for a seedling damping-off caused by Rhizoctonia fungus in tomato, a soil pest control agent having a high gliotoxin production in the evaluation in (3) above was selected. The soil pest control test was conducted. Rhizoctonia bacterium cultured with bran was mixed with sterilized soil [Sakata Seed Co .; Sakata Super Mix] to produce soil contaminated with Rhizoctonia. In this contaminated soil, the soil pest control agents produced in Examples 1, 2 and 4 among the soil pest control agents produced in (2) above were respectively contained at a ratio of 5 v / v.
%, And a section to which no soil pest control agent was added was set, and a plant test was performed on each of these sections using tomato [cultivar: House Momotaro].

Tomato seeds were mixed with a soil pest control agent in contaminated soil, sown 5 days later, and examined 14 days after the sowing date. Then, the number of healthy seedlings and the number of seedlings affected by take-all were examined, and the healthy stock ratio was calculated from these numbers. In addition, an index of the healthy strain rate with respect to the healthy seedling rate in the zone where no soil pest control agent was added was calculated as a non-treatment index. Table 3 shows the results.

[0036]

[Table 3]

(5) Control test of Fusarium lettuce root rot As a soil disease, lettuce root rot caused by Fusarium bacterium was selected from the soil pest control agents having high gliotoxin production in the evaluation in (3) above. The soil pest control test was conducted. Fusarium-contaminated soil collected from a lettuce field in Gunma Prefecture was used as the contaminated soil. As a result of measuring the number of Fusarium bacteria in 1 g of this contaminated soil, the colony forming unit was 3.5 × 10 ⁶ cfu.

In this contaminated soil, of the soil pest control agents produced in the above (2), the soil pest control agents produced in Examples 1, 2 and 4 were respectively contained at 5 v / v%.
And a section to which no soil pest control agent was added was set, and a plant test was performed on each of these sections using lettuce [cultivar: Patriot].

The lettuce seeds were mixed with the soil pest control agent in the contaminated soil, sown 5 days later, and examined three weeks after the sowing date. Then, the number of healthy seedlings and the number of seedlings infected with root rot were examined, and the percentage of healthy seedlings was calculated from these numbers. The index of the ratio of healthy seedlings to the ratio of healthy seedlings in the zone where no soil pest control agent was added was calculated as a non-treatment index. Furthermore, the number of Fusarium bacteria living in the soil three weeks after the seeding date was measured, and an index for no treatment was calculated. The results are shown in Table 4.

[0040]

[Table 4]

Comparative Examples 1 to 3 (1) of Examples 1 to 13
In the evaluation of the known ATCC 31969 strain having the ability to produce relatively large amounts of gliotoxin,
The same evaluation test as in Examples 1 to 13 was performed. (1) Manufacture of soil pest control agent AT instead of Gliocladium virens strain G2
Except for using CC31969 strain, a soil pest control agent was produced in the same manner as in (1) of Examples 1 to 13. The mixing ratio is shown in Table 5.

[0042]

[Table 5]

(2) Test for controlling Rhizoctonia seedling wilt Other than using the soil pest control agent obtained in (1) above,
A control test of Rhizoctonia seedling wilt of tomato was performed in the same manner as in (4) of Examples 1 to 13. These results
It is shown in Table 6.

[0044]

[Table 6]

(3) Test for controlling root rot of Fusarium lettuce Root rot control other than the soil pest control agent obtained in (1) above was used.
A control test of root rot caused by Fusarium bacterium on lettuce was carried out in the same manner as (5) of Examples 1 to 13. The results are shown in Table 7.

[0046]

[Table 7]

[0047]

According to the present invention, it is possible to provide a soil pest control agent and a method for controlling soil pests which are simple, have high safety, and can control soil pests without adversely affecting the natural environment. it can.

Claims (10)

[Claims] 1. Gli cladium berenz (Gli)
ocladiumvirens) G2 [FERM P-
17381] A soil pest control agent containing a culture of a strain. 2. Gliocladium beliens (Gli)
ocladiumvirens) G2 [FERM P-
17381] A soil pest control agent comprising a mixture of a culture of a strain, a bulking agent and a nutrient. 3. The nutrient source is selected from the group consisting of glucose, ammonium sulfate, soy flour, bran and flour.
3. The soil pest control agent according to claim 2, which is a species or two or more species. 4. The soil pest control agent according to claim 1, wherein the soil pest is a fungus, a bacterium, a nematode, a nematode-borne virus or a microorganism-borne virus. 5. Gliocladium beliens (Gli)
ocladiumvirens) G2 [FERM P-
17381] A method for controlling soil pests, which comprises using a culture of a strain. 6. Gliocladium beliens (Gli)
ocladiumvirens) G2 [FERM P-
17381] A method for controlling pests and insect pests, which comprises using a mixture of a culture of a strain, a bulking agent and a nutrient. 7. The nutrient source is selected from the group consisting of glucose, ammonium sulfate, soy flour, bran and flour.
The soil pest control method according to claim 6, wherein the method is a seed or two or more substances. 8. The method according to claim 5, wherein the soil pest is a fungus, a bacterium, a nematode, a nematode-borne virus or a microorganism-borne virus. 9. The method for controlling pests and insect pests according to claim 5, wherein the soil is a field soil before planting or a cultivation of seedlings before transplantation. 10. Gliocladium bilens (Gli)
ocladiumvirens) G2 [FERM P-
17381] strain.