JPH0853317A - Production of soil disease damage controller - Google Patents

Abstract

PURPOSE:To expensively obtain the subject agent containing a culture of a fungus belonging to the genus Gliocladium not containing an antimicrobial substance by maintaining pH of a medium on and after the logarithmic growth phase of culture of the fungus belonging to the genus Gliocladium in neutrality to alkalinity. CONSTITUTION:A medium on and after the logarithmic growth phase of culture of a fungus belonging to the genus Gliocladium is maintained at pH 7-10 (more preferably its retention time is at least 120 minutes) to give a soil disease damage controller mixed with a culture product of the fungus belonging to the genus Gliocladium. Gliocladium virens, Gliocladium catenulatum or Gliocladium deliquescens is preferable as the fungus belonging to the genus Gliocladium. A medium containing a solid component derived from a plant and a porous carrier is preferable as the medium useful for the culture. The soil disease damage controller is inexpensively and simply produced and safely used by this method.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a soil disease control agent for plants and a soil disease control method for plants, and more particularly to a soil for plants containing a culture of a fungus belonging to the genus Gliocladium. The present invention relates to a method for producing a disease control agent.

[0002]

2. Description of the Related Art In recent years, soil diseases of cultivated plants have frequently occurred due to a decrease in the amount of organic fertilizers such as compost used and continuous cultivation of highly profitable crops. As a method of protecting cultivated plants from soil pathogens that cause such soil diseases, a method of sterilizing soil by chemical means has been widely performed, but safety,
There were many problems in terms of environment and sustainability of effects. Therefore, in recent years, in consideration of safety and sustainability of effects, a method of preventing the occurrence of soil diseases by using a microorganism that antagonizes the soil pathogen has been widely used.

Among the fungi used in the biological control method for soil diseases as described above, fungi belonging to the genus Gliocladium are wilt, root rot and stem rot of many plants. It is one of the most promising fungi for antagonizing soil pathogenic fungi belonging to the genus Pythium and the genus Rhizoctonia, which are considered to be the causes.

The mechanism of such disease control by Gliocladium spp., That is, the mechanism of antagonism with the pathogen, is due to the action of antibacterial substances such as gliotoxins produced by Gliocladium spp. It is considered to be due to the action of a lytic enzyme, or due to the fact that gliocladium spp.

However, the above-mentioned antibacterial substance has a problem from the viewpoint of safety, and in the case of producing a culture containing Gliocladium sp. As a soil disease controlling agent, in order to produce a more safe preparation, It is more preferable to remove the antibacterial substance. Therefore, as a method for separating the antibacterial substance produced by Gliocladium spp. From the bacterial cells, a method of separating the bacterial cells from the broth after culture using a centrifuge or a filter can be easily considered. However, treating Gliocladium sp. Cultures using these devices complicates the process and is problematic in terms of cost and labor.

Further, when culturing Gliocladium spp., A method may be considered in which the medium is made alkaline to a pH of 7 or higher to prevent the accumulation of antibacterial substances. Since it is slower than the usual culture, it is inconvenient in terms of productivity.

[0007]

DISCLOSURE OF THE INVENTION The present invention has been made from the above viewpoints, and provides a soil disease control agent containing a fungal culture belonging to the genus Gliocladium that does not contain an antibacterial substance, at low cost and simply. It is an object to provide a manufacturing method.

[0008]

Means for Solving the Problems In order to solve the above-mentioned problems, the present invention provides a method for producing a soil disease controlling agent for plants containing a culture of a fungus belonging to the genus Gliocladium. It was decided that the medium after the logarithmic growth phase of the culture of the fungus belonging to the genus Ocladium should be maintained at pH 7 to 10.

The present invention will be described in detail below.

<1> Fungus belonging to the genus Gliocladium The fungus belonging to the genus Gliocladium used for the production of the soil disease controlling agent of the present invention is particularly limited as long as it is a fungus belonging to the genus Gliocladium. Not, for example, Gliocladium aureum,
Gliocladium
catenulatum), Gliocladium deliquescens, Gliocladium
Nigram (Gliocladium nigram), Gliocladium penicilloides (Gliocladium penicilloides), Gliocladium roseum (Gliocladium roseum),
Gliocladium sa
gariensis), Gliocladium velmoeseni (Gli
ocladium vermoeseni) and Gliocladium virens. These may be used alone or in combination of two or more.

Further, among the fungi belonging to the genus Gliocladium, Gliocladium virens, Gliocladium catenulatum, Gliocladium
Delicate essence is preferably used in the present invention.

<2> Cultivation of fungus belonging to the genus Gliocladium In the present invention, by maintaining the medium at pH 7 to 10 after the logarithmic growth phase of the culture of the fungus belonging to the genus Gliocladium, It is characterized by removing an antibacterial substance in a culture of a genus.

In the present invention, the medium used for culturing the above-mentioned fungus belonging to the genus Gliocladium is not particularly limited, and is a liquid medium or a solid medium usually used for culturing Gliocladium. A medium is used.

As the liquid medium, specifically, a medium containing glucose, saccharose, starch, molasses, etc. as a carbon source and nitrate nitrogen, ammonia nitrogen, urea, peptone, yeast extract etc. as a nitrogen source. Further, if necessary, a trace amount of a metal salt of phosphoric acid, potassium, magnesium or the like can be added thereto. When a liquid medium is used, it may be cultured by an ordinary method using a culture tank or the like.

The solid medium may be a porous carrier such as an inorganic porous carrier such as vermiculite, zeolite, attapulgite, sepialite, montmorillonite, perlite, Kanuma soil, red gemstone, pumice stone, charcoal or coral sand. Examples include those impregnated with a medium.

As a solid medium, plant-derived solid components such as corn residue, sweet potato, potato peel and residue after starch separation, fruit squeezing residue, stevia extract residue, beer residue, liquor residue (including yeast) ), Vegetables (stems, leaves, seeds, etc.), wheat, bran, barley, rye,
Rice, cereals such as rice bran, soybeans, red beans, soybean meal, okara,
Beans such as peanut residue, rapeseed residue, sunflower residue,
Solid components and the like obtained from oil seeds such as cottonseed meal and porous carriers, for example, inorganic porous materials such as zeolite, attapulgite, sepialite, montmorillonite, perlite, Kanuma soil, red clay soil, pumice stone, charcoal and coral sand When used in combination with a carrier or the like, a mixture of these, a medium containing micronutrients such as nitrogen, phosphate, potassium, magnesium, and the like, water, etc., is appropriately added to form gliocladium bacteria. Spores of the
It is preferable in that a soil disease controlling agent having high usability and disease controlling effect can be obtained.

Culturing conditions may be the same as those for culturing Gliocladium spp., Except that the medium is maintained at pH 7 to 10 after the logarithmic growth phase.
The culture temperature is preferably 10 to 45 ° C, more preferably 15 to 35 ° C. About the culture time
It is preferably 40 days, more preferably 3 to 20.
Is the day.

In the present invention, when culturing Gliocladium sp. Using the above medium, the medium is maintained at pH 7 to 10 after the logarithmic growth phase of the culture. Adjust the pH of the medium to 7-1 during the logarithmic growth phase and before.
When kept at 0, the growth of Gliocladium spp. Deteriorates. Therefore, when the medium is prepared, it may be adjusted to a normal pH condition, that is, pH 5 to 7. By making the medium alkaline after the logarithmic growth phase, the antibacterial substance can be removed in the state where the bacterial cells are sufficiently grown. Therefore, in the present invention, "after the logarithmic growth phase" does not mean strictly after the boundary between the logarithmic growth phase and the stationary phase thereafter, and may include the middle phase or the late phase of the logarithmic growth phase. It means that the pH of the medium may be maintained at 7 to 10 as long as the cell amount is obtained.

The culture may be continued while the pH of the medium is kept at 7 to 10, or may be left as it is after the culture is completed. In the flask culture, various buffers such as phosphate buffer, Tris-hydrochloric acid buffer, citrate buffer, glycine-sodium hydroxide buffer, etc. are used for pH adjustment, and in fermenter culture. It is preferable to use an alkali, for example, an aqueous solution of sodium hydroxide, an aqueous solution of potassium hydroxide, aqueous ammonia, or the like. When the culture is solid culture, it is preferable to spray a buffer solution having a pH of 7 to 10 after the culture is completed.

The period for maintaining the medium at pH 7 to 10 is preferably at least 120 minutes. pH 7
If the culture time in the medium of 10 to less than 120 minutes, the antibacterial substance may not be sufficiently decomposed. In addition, this period may be at most about 24 hours, and even if the culture is continued for a longer period of time, the effect reaches the ceiling and is uneconomical. The temperature during this period is not particularly limited, but is preferably 10 to 40 ° C.

During the above culture, it is preferable that the cells of the genus Gliocladium form spores. Examples of the method for promoting the spore formation of Gliocladium spp. Include a method of irradiating with a fluorescent lamp or a weak UV light during or after the culture. Further, the culture medium, by using a solid medium containing a solid component derived from the above-mentioned plant and a porous carrier, by culturing, spore formation is sufficient, and spores are less likely to fall off Gliocladium spp. Can be obtained.

Here, to give a specific example of the culture of Gliocladium spp. In the present invention, first, the Gliocladium spp. Is liquid-cultured in a potato dextrose medium sterilized by an autoclave or the like. Next, yeast extract, glucose is dissolved in water and the medium whose pH is adjusted to acidic with hydrochloric acid or the like is placed in an Erlenmeyer flask or the like, and sterilized by an autoclave, and then Gliocladium spp. After inoculation, the Erlenmeyer flask is placed in an incubator and shake-cultured at 28 ° C. for 4 days. After culturing, add an appropriate amount of sodium hydroxide aqueous solution to this,
The pH is adjusted to alkaline, and the pH is kept constant at any time, and the mixture is incubated for 3 hours on a reciprocal shaker at 5 ° C.

The culture of the fungus belonging to the genus Gliocladium obtained by culturing in this manner is one in which the antibacterial substances have been sufficiently removed.
If necessary, treatments such as stirring and drying are applied to the soil disease controlling agent of the present invention. In the present invention, the preferable soil disease control agent is 10 to 10 ^{11 with} 1 g of the soil disease control agent being a colony forming unit of a fungus belonging to the genus Gliocladium.
It contains CFU.

In addition to the culture of fungi belonging to the genus Gliocladium, the soil disease controlling agent of the present invention also contains a component usually contained in a soil disease controlling agent, such as a nematicide. can do. Further, a soil disease controlling component other than the culture of the fungus belonging to the genus Gliocladium can be blended.

[0025]

EXAMPLES Examples of the present invention will be described below.

[0026]

Examples 1 to 15 Potato dextrose medium obtained by dissolving 4.0 g of potato leachate powder, 20.0 g of glucose, and 15.0 g of agar in 1 L of distilled water by heating and sterilizing with steam at 120 ° C. for 15 minutes. Dispense into 3 petri dishes, and gliocladium vilens (IFO31959), gliocladium catenatorum (IFO3168)
1), Gliocladium Delic Essence (IFO
7062) was inoculated and cultured in an incubator at 26 ° C for 7 days.

A solution prepared by dissolving yeast extract at a rate of 20 g / L and glucose at a rate of 3 g / L in tap water was adjusted to pH 5.6 with hydrochloric acid and then dispensed in 50 mL aliquots into 16 500 mL Sakaguchi flasks. . This was placed in an autoclave and sterilized at 120 ° C. for 15 minutes, and then 12 agar-cultured gliocladium virulence, 2 gliocladium catenulators and 2 gliocladium delicenses each, inoculum And cultured with shaking at 28 ° C. for 4 days. When the pH at the end of the culture was measured, it was found that the culture solution of Gliocladium virens was 6.2, the culture solution of Gliocladium catenatorum was 5.9, and the culture solution of Gliocladium delicense was 6.3. there were.

Then, 1N was added to the above 16 Sakaguchi flasks.
The culture solution was adjusted to various pH values shown in Table 1 by adding a sodium hydroxide aqueous solution, and then placed in a reciprocal shaker at 5 ° C. and incubated for various times shown in Table 1. At this time, the pH of the culture solution was adjusted so as to always be constant while adding an aqueous sodium hydroxide solution.

After the incubation, 1N hydrochloric acid was added to each culture solution to adjust the pH to 6.5, and a filter paper was placed on the Buchner funnel to separate the bacterial cells, and the filtrate was transferred to a separatory funnel and added to acetic acid. 100 mL of an equal volume mixture of ethyl and toluene was added and shaken vigorously to transfer the antibacterial substance in the culture solution to the organic solvent layer. The organic solvent layers in the culture solution were collected, dehydrated by adding anhydrous sodium sulfate, filtered, and the obtained filtrate was concentrated under reduced pressure using a rotary evaporator to obtain a dry solid. The obtained dried solid was dissolved in ethanol and filled up to 5 mL, and the residual antibacterial active substance was measured by the following method.

A 6 mmφ pulp disk was impregnated with 0.3 mL of the organic solvent extract filled up with ethanol, and dried in a clean bench. This was placed in the center of an 8.5 cmφ petri dish containing potato dextrose agar prepared in the same manner as above,
A culture of Pythium ultinum or Rhizoctonia solani, which had been previously cultured in the same potato dextrose agar medium, was punched out with a 9 mmφ cork borer together with the medium, and a cross shape was formed at a position 3 cm from the center. I placed them in four places. This petri dish was placed in an incubator at 25 ° C. and incubated for 6 days, and then taken out and the size of the inhibition circle formed was measured. The results are shown in Table 1.

[0031]

[Table 1]

On the other hand, in Example 11, the mixture of spores and mycelia of Gliocladium virens remaining on the surface of the filter paper of Buchner-Rot was collected and used as it was as a soil disease control agent obtained by the production method of the present invention. The control method for soil diseases was evaluated by the following method.

2 kg of Kuroboku soil was mixed with 3 kg of humus, and this was used as a chemical fertilizer (N: P: K = 10: 10: 10).
10 g was added to prepare a soil. To this soil, the total amount of the soil disease controlling agent (a mixture of spores of glyocladium virens remaining on the surface of the filter paper of Buchner-Rot and mycelia) obtained in Example 11 was added, and mixed sufficiently with stirring. Further, for comparison, a soil prepared by the same method as above was prepared except that the soil disease controlling agent of the present invention was not added.

On the above two types of soils, Verticillium dahliae, a pathogenic bacterium isolated from eggplants affected by half-body wilt, was preliminarily placed on a bran at 25 ° C.
50 g of each pathogen-containing bran obtained by culturing for 7 days was added and mixed.

Each of these soils was packed into 10 plastic pots of No. 5, and after irrigation, they were left in a greenhouse at 25 to 32 ° C. for 4 days. Then, cell-forming seedlings of eggplant (cultivar: Senryo No. 2) were transplanted on the 25th day after sowing, and for 2 days for 40 days.
It was grown in a greenhouse at 5 to 34 ° C, and the number of strains that developed disease during this period was counted.

As a result, for comparison, 3 strains developed in 10 strains of eggplants grown in the soil which was not applied, but the soil disease controlling agent obtained by the production method of the present invention was applied. Among the 10 eggplants grown in the soil, none of the strains became ill.

[0037]

Examples 16 to 17 Gliocladium virulence (IFO9168) was used as a strain of the genus Gliocladium, and the pH of the culture solution at the start of culture was 5.5 (at the end of culture) in culture before logarithmic growth. Had a pH of 6.0)
The experiment was conducted in the same manner as in Examples 1 to 15 except that the temperature during incubation was 25 ° C., and the antibacterial active substance in the obtained culture solution was measured in the same manner. Table 2 shows the results.

[0038]

[Table 2]

As is clear from these results, the gliocladium culture obtained by the method of the present invention is higher than the gliocladium culture treated outside the conditions of the present invention. Contains almost no antibacterial substances,
It can be safely used as a soil disease control agent. Further, the production method of the present invention does not affect the soil disease control action of the gliocladium culture, and the gliocladium culture obtained by the method of the present invention is excellent. It can be seen that it has a soil disease control action.

[0040]

Industrial Applicability According to the method of the present invention, a soil disease control agent containing a fungal culture belonging to the genus Gliocladium containing no antibacterial substance can be easily manufactured at low cost.

Claims (4)

[Claims] 1. A method for producing a soil disease controlling agent for a plant, which contains a culture of a fungus belonging to the genus Gliocladium, wherein a medium after the logarithmic growth phase of the culture of the fungus belonging to the genus Gliocladium is used. A method for producing a soil disease controlling agent, which is characterized in that the pH is maintained at 7 to 10. 2. The holding time for holding the pH at 7 to 10 is at least 120 minutes.
A method for producing the soil disease controlling agent as described. 3. The soil according to claim 1, wherein the fungus belonging to the genus Gliocladium is selected from Gliocladium virens, Gliocladium catenatorum, and Gliocladium delicense. A method for producing a disease control agent. 4. The method for producing a soil control agent according to claim 1, wherein the medium used for culturing the fungus belonging to the genus Gliocladium is a solid component derived from vegetable oil and a porous medium. A method for producing a soil disease controlling agent, which is a medium containing a carrier.