JPH06227929A - Method for producing microorganism herbicide for removing eleocharis kuroguwai ohwi and production medium therefor - Google Patents

Abstract

PURPOSE:To massively produce a microorganism herbicide effective for controlling Eleocharis kuroguwai Ohwi which is a strongly injurious weed in paddy fields. CONSTITUTION:Nimbya scirpicola-K-004 (FERM P-11495) is cultured in a liquid medium at an initial pH of 7.5-10, preferably approximately 8, at 18-35 deg.C, preferably 20-27 deg.C, (for 6-8 days for a vibration culture, or for 4-6 days for an aeration culture), and the produced bacterial cells are fractionated with a homogenizer, a single refiner, etc., to provide the microorganism herbicide for controlling the Nimbya scirpicola-K-004. As the liquid medium, there is used a medium properly containing a carbon source, a nitrogen source, inorganic substances and required growth and production-stimulating substances which can be utilized by the K-004 bacteria, and further validamycin A preferably in a concentration of 10-1000ppm.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for mass-producing a microbial herbicide effective for controlling Eloecharis kuroguwai Ohwi, which is a highly damaging weed in paddy fields, and a production medium therefor.

[0002]

2. Description of the Prior Art A fungus (filamentous fungus) Nimbya scirpicola K-004 (Microtechnological Research Institute No. 11495) isolated from diseased stems of Kurogui is applied to Kurogui, which is a highly damaging weed in paddy fields, to proliferate the host. , A control method for suppressing fertility has been attempted (Japanese Patent Application No. 3-298568).
issue).

When Nimbya scirpicola K-004 is applied to Kurogui , spores of the same bacterium, which are generally excellent in durability such as drought resistance and heat resistance, are applied to Kurogui as the main component of the herbicide. The spores applied to Kurogwai are mainly cultivated and produced by the plate culture method.

[0004]

Although a large amount of spores, which are the main components of microbial herbicides, are required, it is difficult to mass-produce spores by the plate culture method. That is, Nimbya scirpicola K
-004 is about 1 × 10 6 per dish having a diameter of 9 cm on V8 agar or potato dextrose agar plate medium.
^Five spores are formed, and the amount of sporulation is proportional to the surface area of the medium. The production of spores on an industrial scale requires a large area for sporulation, and thus spore production by the plating method is practically difficult.

A liquid culture method is also considered as a culture method, but it is difficult to mass-produce spores by this culture method. That is, the present inventors have found that a liquid medium (Czapek Docks, modified Richard, potato dextrose, oatmeal, V8) commonly used for culturing filamentous fungi.
, Etc.) was examined for spore formation by liquid shaking culture in various media, but no spores were formed in the liquid media.

On the other hand, the present inventors have discovered that the filamentous hyphae of the same fungus obtained by liquid shaking culture in a flask have the same pathogenicity as spores. However, in the case of microbial cell growth by liquid culture, a microbial cell mass is usually formed, and thus there is a problem in ease of use as a herbicide. The present invention is to increase the yield of cells that are the main components of herbicides in liquid culture, fragment the cell mass using a crushing device, and the number of mycelium fragments that can grow on the medium thereby (Colony Forming Units, hereinafter " CF
U ”. ), Homogenization of the culture product, and fragmentation of the filamentous mycelium to improve the ease of handling in the subsequent steps such as formulation and application after culture production.

[0007]

The present invention includes the following inventions. (1) A method for producing a microbial herbicide for controlling Klogwai , which comprises culturing Nimbya scirpicola K-004 in a liquid medium and fragmenting the obtained mycelium. (2) The production method according to (1) above, wherein the initial pH of the medium is set to 7.5 to 10. (3) The production method according to (1) or (2) above, wherein a medium containing validamycin A is used as the liquid medium. (4) Nimbya which comprises validamycin A
Production medium for mycelium of scirpicola K-004.

Nimbya scirpicola K-0 used in the present invention
04 (hereinafter referred to as "K-004 bacterium") has been deposited at the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology as Micromachine Research Institute No. 11495. As the medium, either synthetic medium or natural medium can be used as long as it contains a carbon source, a nitrogen source, an inorganic substance which can be assimilated by K-004 bacterium, an inorganic substance and necessary growth and production promoting substances.

As the carbon source, for example, glucose, sucrose, galactose, maltose, trehalose, xylose, dextrin, starch, fructose, glycerol, lactose, mannose, arabinose, mannitol, molasses, etc. may be used alone or in combination. , Sucrose, galactose, maltose, trehalose, xylose, dextrin, corn starch, soluble starch, and fructose are preferably used alone or in combination.
The carbon source concentration in the medium is preferably 70 to 100 g / l
Is.

As the nitrogen source, for example, ammonium chloride, ammonium nitrate, sodium nitrate, ammonium sulfate, urea, casein, peptone, meat extract, yeast extract, dried yeast, corn steep liquor, soybean powder, casamino acid, etc., alone or in combination. However, sodium nitrate, casein, peptone, and yeast extract are preferably used alone or in combination.

It is preferable to add validamycin A to the medium. The validamycin A concentration in the medium at this time is preferably 10 to 1000 ppm. In addition, inorganic salts such as sodium chloride, potassium chloride, magnesium sulfate, calcium carbonate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ferrous sulfate, calcium chloride, manganese sulfate, zinc sulfate, and copper sulfate may be added as necessary. Add. Furthermore, a trace component that promotes the growth of K-004 can be appropriately added.

Cultivation is usually carried out at 18 to 18 under appropriate aerobic conditions.
It is carried out at 35 ° C, preferably 20 to 27 ° C. The medium has an initial pH of 7.5-10, preferably about 8. During the culture, it is preferable to add no acid or alkali and keep the pH at a natural level. The culture method may be shaking culture or aeration culture by jar fermenter or tank culture, as long as a liquid medium is used.

When the culture is carried out under the above-mentioned conditions for 6 to 8 days in the shaking culture and 4 to 6 days in the aeration culture, the cells grow efficiently. After production of the mycelium, it is preferable to fragment the mycelium with a device for fragmenting the mycelium, such as a homogenizer or a single disc refiner. By this fragmentation treatment, homogenization of products, CFU, and increase of infection source are achieved. For example, a homogenizer (Silverson
The crushing time when using L4R type) is preferably 1
0 seconds to 3 minutes.

[0014]

EXAMPLES The present invention will be described in more detail with reference to test examples and examples, but the scope of the present invention is not limited thereto. (Test Example 1) Carbon source in medium Czapek-Dox liquid medium containing 1 g / l of yeast extract was used as a basic medium. The basal medium was inoculated with the K-004 bacterium, and the mixture was subjected to liquid shaking culture at 25 ° C. using a 500 ml Erlenmeyer flask. As the carbon source in the basic medium, 12 kinds of carbon sources shown in Table 1 were used, and the carbon source concentration in the medium was 3
The initial pH of the medium was set to 6 with 0 g / l. The cell growth on the 14th day after the start of culture was compared. The results are shown in Table 1.

[0015]

[Table 1] K-004 fungus utilized all the carbon sources used in the test.
Among them, glucose, sucrose, galactose,
It grew well in liquid medium using maltose, fructose, trehalose, xylose, dextrin, soluble starch and corn starch as carbon sources.

Test Example 2 Glucose Concentration in Medium Czapek-Dox liquid medium supplemented with 1 g / l of yeast extract was used as the basic medium. K-004 strains were subjected to liquid shaking culture using a liquid medium in which the carbon source (glucose) concentration in the basal medium was set to 20, 50, 60, 70, and 100 g / l, respectively. The dry yield of the bacterial cells grown on the 14th day after the start of culture was measured. The results are shown in Table 2. The initial pH of the medium was set to 6. In Table 2, the dry yield of cells obtained when culturing at a glucose concentration of 20 g / l was 10
It was set to 0.

[0017]

[Table 2] K-004 using liquid medium containing high concentration of glucose
The dry yield of the cells increased when the cells were cultured. The maximum dry cell yield was 100 g / glucose in the culture solution.
It was recognized when it was l. (Test Example 3) Nitrogen source in medium For comparison of nitrogen sources in the Tzapek-Dox basal medium, liquid culture was tried in a liquid medium using 6 kinds of nitrogen sources shown in Table 3. The initial pH of the medium was set to 6.
14 days after the start of culture, the yield of bacterial cells was measured and compared. The results are shown in Table 3.

[0018]

[Table 3] K-004 fungi utilized all the nitrogen sources used in the test.
Among them, when sodium nitrate, yeast extract, casein and peptone were used as nitrogen sources, the bacterial cells were obtained in high yield. Test Example 4 Temperature during Culture and Yield of Producing Bacteria Potato dextrose liquid medium was used as a medium, and the initial pH of the medium was 5.6. K-004 bacteria 12,
After culturing at 18, 20, 24, 27, 30, and 35 ° C., respectively, the dry yields of the grown bacterial cells were compared. The results are shown in Table 4. In Table 4, the dry yield of cells obtained by the culture at 27 ° C. was set to 100.

[0019]

[Table 4] The cell culture production of K-004 can be performed at 18 to 35 ° C, and the cells were produced at a high yield by preferably culturing at around 20 to 27 ° C. (Example 1) Effect of addition of validamycin A To the C100 medium shown in Table 5, 10 to 4 validamycin A was added.
The initial pH was set to 8 in a liquid medium containing 000 ppm, and K-004 bacteria were subjected to liquid shaking culture at 25 ° C for 6 days, and 100 ml of the obtained culture solution containing a cell mass was homogenized (Silverson L4R type) for 2 minutes. It was fragmented by crushing, and CFU was compared. The results are shown in Table 6. In Table 6, CFU of the group without addition of validamycin A was set to 100.

[0020]

[Table 5]

[0021]

[Table 6] From Table 6, it can be seen that the addition of validamycin A to the medium at 10 to 1000 ppm increases the CFU in the medium. CFU was the highest in the culture in the medium containing 1000 ppm, and the CFU was increased 5.5 times as compared with the case where no validamycin A was added.

(Example 2) Effect of pH of culture medium C100 culture medium shown in Table 5 was treated with 1000 mg of validamycin A.
The initial pH of the liquid medium added with ppm was set to 3 to 10, and K-004 bacteria were cultured at 25 ° C. for 6 days with liquid shaking.
CFU was compared by fragmenting by crushing 100 ml of the obtained culture solution containing the cell mass with a homogenizer (Silverson L4R type) for 2 minutes. The results are shown in Table 7. In Table 7, CFU when using a liquid medium in which the initial pH was set to 6
Was set to 100.

[0023]

[Table 7] When the initial pH was set to 8, the CFU after crushing the bacterial cells increased the most, and increased about 3 times as compared with the case of the initial pH 6. From this, it can be seen that CFU significantly increases by setting the initial pH to 7.5 to 10. (Example 3) Increase in CFU due to fragmentation of mycelium 1000 mg of validamycin A was added to C100 medium shown in Table 5.
The initial pH of the liquid medium added with ppm was set to 8 and K-
The 004 strain was subjected to liquid shaking culture at 25 ° C. for 6 days, and the obtained cultured bacterial cells were fragmented using a homogenizer (Silverson L4R type). Table 8 shows the relationship between the treatment time of the homogenizer and the CFU. In Table 8, the CFU obtained by the homogenizer treatment for 10 seconds was set to 100.

[0024]

[Table 8] CFU by homogenizer treatment for 10 seconds to 3 minutes
It was confirmed that the fragmentation of mycelium is a very effective means for increasing CFU, since the number was significantly increased 100-fold. (Example 4) Production of mycelia by tank culture and liquid shaking culture 1000 C of the C100 medium shown in Table 5 was treated with 1000 mg of validamycin A.
The initial pH of the liquid medium added with ppm was set to 8 and 10
While stirring with aeration at 0 rpm, 0.5 vvm, 25 ° C,
K-004 was cultured in a 50-liter tank for 5 days. The obtained bacterial cells were separated by a centrifuge (may be separated from the culture filtrate by filtration). Further, liquid shaking culture was carried out for 6 days under the same medium, pH and temperature conditions. The results are shown in Table 9.

[0025]

[Table 9] From the above results, it can be seen that the target bacterial cells can be mass-produced by culturing the K-004 bacterium by the production method of the present invention. Particularly, the tank culture increases the productivity of the bacterial cells, and the bacterial cells can be efficiently produced. (Example 5) Herbicidal activity of bacterial cells cultivated by various production methods K-004 spores obtained by V8 agar plate culture and liquid shaking culture of Example 4 and K-004 obtained by 50-liter tank culture. The herbicidal activity was compared by applying the mycelium of the above to the kurogwai which is growing in the greenhouse. <Preparation of Spore Suspension and Inoculation Conditions> After spraying the spore suspension onto the crocodile by air spray, it was immediately placed in a humid chamber at 25 ° C. for 24 hours and observed in a greenhouse at 25 ° C. <Mycelium preparation and inoculation conditions> The mycelium was homogenized with a homogenizer (Silverso) in both liquid shaking culture and 50-liter tank culture.
n L4R type) was crushed for 2 minutes to fragment. The cells were resuspended in C100 medium shown in Table 5 and spray-inoculated on Kurogui. Put the inoculated Kurowai in a greenhouse at 25 ° C for 24 hours.
Leave for 24 hours in a humid chamber at 25 ° C, then repeat for 2 hours.
It was returned to the greenhouse at 5 ° C.

Two weeks after the inoculation, the symptom of kurogwai was observed and the herbicidal effect was investigated. The results are shown in Table 10.

[0027]

[Table 10] In Table 10, the herbicidal activity was 0 in the non-treated cells, 25 in the case where the plant growth was 3/4 compared to the non-treated cells,
The average value was obtained by making a judgment based on a conservative view, with the case of 1/2 being 50, the case of 1/4 being 75, and plant death being 100. From Table 10, it is understood that the herbicidal activity of the bacterial cells obtained by the tank culture has the same herbicidal activity as the spores obtained by the plate culture method and the bacterial cells obtained by the shaking culture method.

[0028]

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to efficiently produce a microbial herbicide for controlling kurogowai which comprises filamentous hyphae or fragments thereof and has a high CFU.

Claims (4)

[Claims] 1. A method for producing a microbial herbicide for controlling Klogui , which comprises culturing Nimbya scirpicola K-004 in a liquid medium and fragmenting the obtained mycelium. 2. The method according to claim 1, wherein the initial pH of the medium is set to 7.5-10. 3. The production method according to claim 1, wherein a medium containing validamycin A is used as the liquid medium. 4. A production medium for the mycelium of Nimbya scirpicola K-004, which contains validamycin A.