JPH10146185A - Heat-resistant bacillus, lawn grass pathogenic bacterium control agent containing the bacillus, organic fertilizer, and their production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new heat-resistant bacterium comprising heat-resistant Bacillus subtilis or Bacillus firmus obtained by the treatment of sewage sludges in an autoclave and capable of inhibiting the growth of lawn grass pathogenic bacteria, and useful for lawn grass pathogenic bacterium control agents, organic fertilizers, etc. SOLUTION: This new heat-resistant Bacillus subtilis AS-2-1 (FERM P-15797) or Bacillus firmus AS-2-2 (FERM P-15798) capable of inhibiting the growth of lawn grass pathogenic bacteria is obtained by treating sewage sludges in an autoclave. The spore- formed culture products of the bacteria are useful for lawn grass pathogenic bacterium control agents exhibiting high control effects in small use amounts, and also for organic fertilizers capable of healthily growing lawn grasses and maintaining the green color of the lawn grasses for long periods. The heat-resistant bacteria are obtained by treating the sewage sludges in an autoclave, culturing the treated sludges, and subsequently selecting the lawn grass growth-inhibiting strain from the spore-formed cultured product by a screening method comprising culturing the cultured product together lawn grass pathogenic bacteria.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a heat-resistant bacillus that suppresses the growth of turfgrass pathogens, a turfgrass fungus controlling agent and an organic fertilizer containing the bacillus as an active ingredient, and methods for producing the same. More specifically, a turfgrass fungus-controlling agent containing the above-mentioned heat-resistant bacillus, which suppresses the occurrence of disease on turfgrass and can control the generated disease, an organic fertilizer that can also grow turfgrass healthy and control disease, and the like. Related to the manufacture of.

[0002]

2. Description of the Related Art As means for controlling turfgrass diseases at golf courses, there are cultivation control and pesticide control. Most of the golf courses use pesticides. On the other hand, sewage sludge has recently been used in compost as a soil-improving material in green agricultural lands such as golf courses and fields for recycling of resources. Sewage sludge has also been studied for use as a construction material, such as geological improvement materials, road pavement materials, concrete aggregates, porcelain tubes, bricks, and tiles. Have been.

[0003]

In a golf course, turfgrass is frequently trimmed because the beauty of the turfgrass is a criterion for determining the quality of the golf course. This pruning causes damage to the turfgrass tissue (leaves, stems, rhizomes, etc.), which serves as a path for pathogens and makes the disease more likely to occur. At golf courses, pesticides are sprayed to maintain the beauty of the turfgrass and to prevent the spread of the disease. Therefore, a golf course tends to use an excessive amount of pesticide, and there is a problem that the applied pesticide causes environmental pollution and water pollution. Due to such pollution problems, in recent years, there have been campaigns against golf course construction and environmental protection in various places.

[0004] Regarding the use of pesticides in golf courses, the "Guidelines for Safe Use of Pesticides for Golf Courses" has been issued from prefectures to golf courses, and the total amount of pesticides used has been regulated. Therefore, it is anticipated that regulations on the use of pesticides will become increasingly strict in the future. Therefore, development of a different type of pesticide than conventionally used is desired. In other words, there has been a demand for pesticides that have a high effect on pathogenic bacteria with a small amount of use and do not cause environmental pollution or water pollution.

[0005] As this type of pesticide, there are pesticides that use microorganisms. When microorganisms are used as pesticides, two requirements are required. One is the prescription (fo
rmulation) until the final application (from 6 months to 2 years if possible) is to maintain a stable potency and microbial load, and the other is that the microorganisms contained in the product can be transported or stored during the transportation of the product. It can withstand high temperatures (50 ° C to 60 ° C). Conventionally, no pesticides satisfying such requirements existed.

[0006]

Means for Solving the Problems The inventors of the present invention have intensively studied to solve the above problems, and the occurrence of turfgrass disease at a golf course where sewage sludge compost is continuously used has been extremely high. We paid attention to few phenomena. And, from the sewage sludge compost, heat-resistant microorganisms showing an antagonistic effect against turfgrass pathogens were separated, and they were found to be useful as turfgrass pathogen control and turfgrass organic fertilizers, and completed the present invention. It is.

The present invention is a bacterium belonging to the genus Bacillus, which has heat resistance obtained by screening from sewage sludge and has a strong antagonistic power against pathogens of turfgrass, and for controlling disease of turfgrass containing a culture of this fungus. An object of the present invention is to provide a lawn grass pathogen control agent and an organic fertilizer comprising a coating containing the cells and sewage sludge. Another object of the present invention is to provide a method for producing the turfgrass pathogen control agent and the organic fertilizer.

That is, a first aspect of the present invention is a heat-resistant Bacillus subtilis or Bacillus famas which suppresses the growth of turfgrass pathogens obtained by autoclaving sewage sludge. Here, the autoclave treatment is performed by
It is sterilized at 20 ° C for 20 minutes.

A second aspect of the present invention is a turfgrass pathogen-controlling agent comprising the above-mentioned spore-forming culture of heat-resistant Bacillus subtilis and / or Bacillus famas solely and / or as a mixture. . The spore-forming culture is incubated at 25 ° C to 30 ° C for 2-3 days, then at 55 ° C
It is obtained by culturing at 65 ° C. for 2 to 3 days.

[0010] A third aspect of the present invention comprises a step of autoclaving the sewage sludge compost, a step of screening heat-resistant Bacillus subtilis and / or Bacillus famas from the autoclaved sewage sludge compost, Forming a spore-forming culture by forming and culturing the spore-forming culture;
A method for producing a turfgrass pathogen-controlling agent comprising the steps of mixing at a ratio of 〜1: 1 and adsorbing the base at 2 to 8 × 10 ⁸ cfu / g, and crushing the base.

A fourth aspect of the present invention is an organic fertilizer containing 4 to 8% by weight of nitrogen (N): phosphoric acid (P): potassium (K) and sewage sludge. The organic fertilizer may further comprise a coating layer comprising the spore-forming culture of Bacillus subtilis and / or Bacillus famas. The coating layer is characterized by containing a spore-forming culture of the above bacterium, alone or as a mixture, together with a viscous polysaccharide. Further, the viscous polysaccharide is characterized in that it is selected from the group consisting of tragacanth gum, galacturonic acid, fucose, and gum arabic.

[0012] Further, the coating layer comprises the spore-forming culture and the viscous polysaccharide in a ratio of 500: 1 to 1000: 1.
Characterized in that they are mixed with each other. In a fifth embodiment of the present invention, nitrogen (N), phosphoric acid (P), and potassium (K) are added to sewage sludge in a ratio of 4 to 8% by weight and 5: 5: 5 to form a mixture. And a step of preparing a coating solution comprising the spore-forming culture of Bacillus subtilis and / or Bacillus famas and a viscous polysaccharide, and a step of coating the mixture with the coating solution. A method for producing an organic fertilizer, characterized in that: The spore-forming culture is a single culture of the above bacterium or a mixture of the single cultures mixed at a ratio of 7: 3 to 1: 1.

[0013] The viscous polysaccharide used herein is characterized in that it is selected from the group consisting of tragacanth gum, galacturonic acid, fucose, and gum arabic. Further, the mixing ratio of the spore-forming culture and the viscous polysaccharide is 500: 1 to 1000: 1.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION Bacillus subtilis, which is a heat-resistant bacterium belonging to the genus Bacillus, and / or
Alternatively, Bacillus famas is derived from sewage sludge. As the sewage sludge, fermented sewage sludge may be collected and used, or commercially available sewage sludge compost may be used. Examples of commercially available products include Higashi Organic (made by Higashi Sangyo Co., Ltd.) and Tateyama Yuki (made by Tateyama Engineering Co., Ltd.).

The heat-resistant Bacillus subtilis and / or Bacillus famas of the present invention can be obtained by screening the sewage sludge compost after autoclaving and then screening for a bacterium belonging to the genus Bacillus which is resistant to high temperatures (heat resistance). Bacillus). Because these bacteria are heat resistant, they can withstand temperatures of 50-60 ° C. Bacteria belonging to the genus Bacillus form spores as a durable body when the nutritional state is inappropriate, and these spores are extremely resistant to the external environment such as heat, dryness, and drugs. It has been known.

Therefore, when a turfgrass pathogen control agent or an organic fertilizer is manufactured as described below, even when these are stored or transported, when the temperature described above is reached, the bacteria contained in the product are reduced and killed. And the effect is not impaired.

The heat-resistant Bacillus bacterium of the present invention can be obtained by screening by the following method. That is, sludge is collected from sewage sludge during fermentation, air-dried, and sieved to collect sludge having a particle size of about 5 mm. Alternatively, a commercially available sewage sludge compost having a particle size of about 5 mm is used.

The sewage sludge or the sewage sludge compost is appropriately placed in an appropriately-sized Erlenmeyer flask,
Seal with aluminum foil and sterilize by autoclave at 121 ° C for about 20 minutes. Separately, a sterile petri dish containing a nutrient agar medium is prepared. Place the sewage sludge or sewage sludge compost particles cooled after autoclaving on this nutrient agar medium,
Culture in a thermostat at 30 ° C and 55 ° C for 168 to 240 hours to separate the grown microorganisms.

Next, the obtained microorganisms are examined for antagonistic activity against the following turfgrass pathogens. The turfgrass pathogens used were large patch disease (Rhizoctonia solani AG-2-2 (IV)),
Brown patch fungus (Rhizoctonia solani AG-2-2 (II
IB)), Yellow patch disease fungus (binucleate Rhizoctoni)
a AG-D), Pythium aphanidermatum,
Dollar spot disease (Sclerotinia homoeocarpa).

The large patch disease fungus (Rhizoctonia solani)
When AG-2-2 (IV)) infects the turf of a golf course, the foliage of the turf is invaded and becomes water-soaked. Form a ~ 5 cm or 10 m patch. For this reason, infections caused by this fungus are called large patch disease.

The brown patch fungus (Rhizoctonia solan)
When i AG-2-2 (IIIB)) infects the turf of a golf course, the turf creates a cloudy yellow-brown spot, and the whole leaf turns from purple-green to brown, and then the leaf wilts down. And form a patch of light brown, 10-15 cm in diameter, and 50-60 cm when spread. For this reason, infections caused by this bacterium are called Brown Patch Disease.

[0022] Large patch disease and Brown patch disease are
All are soil filamentous fungi, and are caused by Rhizoctonia solani, which causes blight and stem rot of other plants. When yellow patch disease (binucleate Rhizoctonia AG-D) infects turf on a golf course, the turf turns light brown, reddish brown or yellow, forming rings and patches. For this reason, infectious diseases caused by this bacterium are called yellow patch disease.

[0023] Yellow patch disease is a bacterium belonging to the genus Rhizoctonia, but is caused by bimuculate rhizoctonia having two nuclei in mycelial cells. Pythium a
When phanidermatum) infects the turf of a golf course, the turf forms a grayish-brown lesion with a diameter of 10 to 30 cm, but the remaining turf buds recover and turn into a donut-shaped reddish-brown, It becomes reddish brown. For this reason, the infection caused by this fungus is called red blight. Pythiu Aphanidermatum (Pythiu
maphanidermatum) is also the causative agent of tomato cotton rot.

Dollar spot disease (Sclerotinia homoe)
When ocarpa) infects the grass of a golf course, the leaves of the grass become yellow-green soaked water, and then the whole leaf becomes straw-colored. For this reason, infections caused by this fungus are called Dollar spot disease. Sclerotinia homoecarpa is a bacterium belonging to the genus Sclerotinia, and is also a causative fungus of the same genus as the sclerotium fungi of the Solanaceae family.

The antagonism of these pathogens is investigated by confronting culture. That is, two types of bacteria are simultaneously inoculated and cultured on a flat solid medium in a Petri dish with a slight separation. These strains were fixed as in Example 2. The bacteriological properties of the two strains which showed particularly strong antagonistic power are shown in Table 1 below.

[0026]

[Table 1]

The identification was carried out in Bergey's Manual and Cowan an
d Performed according to Steel's. Based on the above mycological properties, these two strains are Bacillus subtilus
s, identification No.AS-2-1) and Bacillus famas
us firmus, AS-2-2). These were added in August 1996.
Deposited with the Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry on March 27, and given accession numbers FERM P-15797 (AS-2-1) and FERM P-15798 (AS-2-2) .

The above-mentioned heat-resistant Bacillus is formulated in a formula
In the case of a turfgrass pathogen controlling agent which requires a long period of time of about 6 months to 2 years from the time of ation) to the final application, it is desirable to form a spore-forming culture by culturing the spore-forming culture. By forming spores, it is possible to stably maintain the efficacy and the amount of bacteria until the time of application.

In order to form spores, the genus Bacillus is cultured in a liquid medium at an appropriate temperature for a certain period of time.
Examples of the medium to be used include a nutrient medium, a yeast extract / albumin medium, and a peptone medium.The nutrient medium is used because the pH of the medium during culture can be maintained from neutral to slightly alkaline. Is preferred. The above bacteria form spores by aerobically culturing at about 20 to 30 ° C. for several days.
Aerobic culture is performed by shaking culture using a shaker,
Alternatively, when a jar fermenter or the like is used, the stirring is performed while blowing air. Depending on culture conditions, static culture may be further performed after shaking culture or the like.

Specifically, the cells are cultured with shaking at 30 ° C. for 6 to 7 days, or with shaking at 30 ° C. for 3 days, and further cultured at 60 ° C. for 2 days as described later. Culturing at 60 ° C. may be performed by any method of static culture and shaking culture. The spore-forming culture obtained by shaking culture at 30 ° C. for 6 to 7 days, alone or as a mixture, is mixed with a base in a predetermined amount to prepare a turfgrass pathogen control agent. When a spore-forming culture obtained by culturing the heat-resistant Bacillus subtilis or Bacillus famas is used as a mixture, it is preferable to mix each culture at a ratio of 7: 3 to 1: 1. . When a 1: 1 mixture is used, the above-mentioned turfgrass pathogenic fungus is highly effective in controlling.

Examples of the base include minerals such as white clay, kaolin, diatomaceous earth, bentonite and zeolite. Because absorption retention of water and nutrients is good,
It is preferable to use diatomaceous earth, kaolin, and zeolite. When diatomaceous earth is used, the effect of preventing caking is high. When the spore-forming culture is mixed with a base, the number of spores to be mixed is not particularly limited. However, culture 10-
When 15 parts by weight and 85-90 parts by weight of the base are mixed, 2-8
× 10 ⁸ cfu / g A composition containing a spore of the base can be obtained.

The above-mentioned heat-resistant Bacillus spp. Was cultured with shaking in a nutrient liquid medium at 30 ° C. for 3 days.
This culture was inoculated on a different substrate than the one described above, and 55
Spores can also be formed by culturing at 6565 ° C. for another 2 to 4 days. Examples of the base used herein include sterilized rice bran, bran, and oat grains. Use of sterilized rice bran is preferable because it is inexpensive and easily available. Inoculation amount is the weight ratio (w / w) with respect to the above-mentioned base.
If it is 10-20%, a composition of 1-5 × 10 ⁹ cfu / g base can be obtained. The culture temperature of 55-65 ° C
This is for promoting the formation of spores and preventing contamination of various bacteria.

The composition thus obtained is air-dried at a temperature of 80 ° C. or less and pulverized to prepare a lawn disease controlling agent. 80
The purpose of air drying at a temperature of not more than ° C is to prevent the aforementioned bacteria from being reduced or killed. For the pulverization, a pot mill, a Waring blender, a homogenizer or the like can be used. In order to obtain fine powder, it is preferable to use a pot mill. Further, by uniformly pulverizing, the Bacillus bacterium contained in the present control agent can be evenly sprayed, so that the control effect against turfgrass disease is enhanced.

The application rate of the turfgrass pathogen-controlling agent containing the bacterium belonging to the genus Bacillus as an active ingredient is usually from 5 to 15%.
g is added to 1 L of water and this is preferably sprayed per m ² . The control agent of the present invention usually exerts a preventive effect when applied before the onset of the disease caused by the above-mentioned pathogenic bacteria. In addition, even when sprayed after the onset of the disease, the spread of the disease can be prevented, and the control effect is further exhibited.

The organic fertilizer of the present invention contains nitrogen fertilizer, phosphate fertilizer and potassium fertilizer as essential components in sewage sludge. The content of the sewage sludge in the organic fertilizer of the present invention,
It is preferable that the amount is 40 parts by weight or more from the viewpoint of the content of organic substances and the fact that the fertilizer becomes a slow-release fertilizer.

Examples of the nitrogen fertilizer include urea, ammonium sulfate, ammonium chloride, ammonium nitrate, monoammonium phosphate and diammonium phosphate. Use of urea, ammonium nitrate, and ammonium chloride is preferable because of a high content of nitrogen, and it is more preferable to use urea. The mixing amount of the nitrogen fertilizer is preferably such that the nitrogen component content is 4 to 8% by weight of the organic fertilizer of the present invention. When the content is 5% by weight, the components can be easily calculated when used.

Examples of the above-mentioned phosphate fertilizer include lime heavy perphosphate, magnesia perphosphoric acid, molten phosphorus fertilizer, heavy clay burnt phosphorus, monopotassium phosphate, dipotassium phosphate and the like. It is preferable to use lime superphosphate, lime heavy phosphate, or the like because it contains a large amount of water-soluble phosphoric acid, and it is more preferable to use lime superphosphate. The mixing amount of the phosphate fertilizer is preferably such that the component content of the phosphoric acid is 4 to 8% by weight of the organic fertilizer of the present invention. 5
When the weight% is used, the components are easy to calculate when used.

[0038] Potassium fertilizers include potash fertilizers used for general agriculture, such as potassium chloride, potassium sulfate, and the like.
Potassium sulfate, potassium nitrate, potassium silicate and the like can be mentioned. It is preferable to use potassium chloride or potassium sulfate because it contains a large amount of potassium and is easily soluble in water and has a quick effect. It is more preferable to use potassium chloride.

The amount of the potassium fertilizer to be mixed is preferably such that the content of potassium is 4 to 8% by weight of the organic fertilizer of the present invention. When the content is 5% by weight, the components can be easily calculated when used. The above-mentioned organic fertilizer, similarly to the production of known organic fertilizers, nitrogen, phosphoric acid and potassium components to the sewage sludge, nitrogen fertilizer, phosphate fertilizer, potassium fertilizer appropriately added, mixed, It is granulated, dried and manufactured.

The application rate of the organic fertilizer of the present invention varies depending on the type of turfgrass, the cultivation place, and the time of spraying, and can be appropriately adjusted, similarly to the fertilizer used for ordinary turfgrass. As a rough guide, it may be about 50 to 150 g / m ^{2 for} tea grounds, fairways and roughs, and about 20 to 50 g / m ² for greens or nurseries.

The above-mentioned organic fertilizer may further have a coating layer containing a spore culture of the heat-resistant bacillus of the genus Bacillus of the present invention. It may contain a viscous polysaccharide. By adding such a viscous polysaccharide, the bacterium of the genus Bacillus of the present invention can be more firmly adhered to the organic fertilizer particles in the organic fertilizer, and the effect of the organic fertilizer can be improved for one year or more. Over a long period of time. Specific examples include uronic acid, galacturonic acid, fucose, galactose, arabinose, rhamnose, chondroitic acid, hyaluronic acid, proteoglycan, tragacanth gum, gum arabic, and CMC.

Because of good adhesiveness, tragacanth rubber,
It is preferable to use galacturonic acid, fucose, gum arabic and the like, and tragacanth rubber is more preferable because it has the highest tackiness. The amount of the above-mentioned viscous polysaccharide to be added varies depending on the kind thereof, but it is usually 0.5 to 5.0 parts per 1000 parts by weight of the spore-forming culture of the heat-resistant Bacillus bacterium of the present invention. And preferably 1 to 2 parts by weight.

The above-mentioned organic fertilizer is prepared by mixing sewage sludge with each component of nitrogen, phosphoric acid and potassium at a predetermined ratio, pulverizing, granulating, drying and sieving. Coating. For this coating, various methods generally used for coating a granular substance can be used. For example, immersion,
Spraying and the like can be mentioned, but it is preferable to perform the spraying method because the water content of the organic fertilizer can be easily adjusted.

More specifically, the coating is performed as follows when the temperature is reduced to 80 ° C. or less in the above-mentioned drying process or after the drying. The amount of the spore-forming liquid culture of the heat-resistant bacterium belonging to the genus Bacillus used for coating is 5 to 15 parts by weight, preferably 10 parts by weight, based on 100 parts by weight of the organic fertilizer. If the amount is less than 5 parts by weight, there is a possibility that all of the particles of the heat-resistant Bacillus bacterium and the organic fertilizer may not adhere to the particles. Because it will happen. Spray the coating solution evenly while mixing the organic fertilizer.

As described above, an organic fertilizer that can grow turfgrass in a healthy manner can be obtained. In addition, since the organic fertilizer contains the spore-forming culture of the heat-resistant Bacillus bacterium of the present invention capable of controlling turfgrass pathogens in the coating layer, it also has an action of controlling turfgrass pathogens. Furthermore, the organic fertilizer containing the coating layer thus obtained can stably maintain the amount of heat-resistant bacteria of the genus Bacillus of the present invention and / or the ability to control pathogenic bacteria for one year or more. .

The application rates in the case of using an organic fertilizer for the control of lawn grass pathogens present invention, as a fertilizer component necessary turfgrass growth, 30 to 150 g / m ^2, preferably from 50 to 100 g / m ^2. The frequency of application may be sprayed once in autumn and once before the onset of illness in early spring. By this application, the occurrence of illness on turfgrass can be suppressed and the turfgrass can be grown healthy. When sprayed in the spring, it is effective in preventing rhizoctonia spring baldness, in which the lawn becomes thin or flaking due to the above-mentioned Rhizoctonia infection.

[0047]

EXAMPLES Next, the present invention will be described more specifically based on the following examples, but the present invention is not limited to these examples. (Example 1) Screening of heat-resistant bacteria from sewage sludge compost A commercially available sewage sludge compost having a particle size of about 5 mm (trade name: Higashi Organic, manufactured by Higashi Sangyo Co., Ltd.) was placed in a 100 ml Erlenmeyer flask.
Add 50ml. The Erlenmeyer flask was covered with aluminum foil and sterilized in an autoclave at 121 ° C. for 20 minutes. In order to screen for bacteria from the sewage sludge compost, a nutrient agar medium and a slant medium of PDA (potato decoction agar medium with glucose added) were respectively prepared according to a conventional method.

[0048] On this agar medium, cooled sewage sludge compost particles were inoculated, and cultured for one week in an incubator at 30 ° C and 55 ° C. The microorganisms that appeared on the agar medium from the particles of the sewage sludge compost were transplanted to the PDA slant medium, and cultured again at 55 ° C. for about one week. As a result, eight strains of heat-resistant bacteria were obtained from the 500 sewage sludge compost particles.

Example 2 Test of Antagonism of Thermostable Bacteria against Turf Grass Pathogens The eight thermostable bacteria obtained in Example 1 are shown in Table 2 below.
The turfgrass shown in Table 3 was tested for antagonistic activity against five strains of pathogenic bacteria.

[0050]

[Table 2]

In the test for antagonistic force, one loopful of the heat-resistant bacterium was inoculated in a liquid nutrient medium at 30 ° C.
After shaking culture for 7 days or shaking culture at 30 ° C. for 3 days, static culture was further performed at 60 ° C. for 2 days to obtain a spore-forming culture. 0.2 mL of the sterilized spore-forming culture was infiltrated into an 8 mm-diameter filter paper disc (trade name: PAPER DISK, manufactured by Toyo Roshi Kaisha, Ltd.), and the disc was agar-cultured (PDA).
Medium) and cultivated confronting turfgrass pathogens shown in Table 2. The temperature of confronting culture varies depending on the type of pathogenic bacterium, but was set to a temperature between 20 and 30 ° C.

The antagonism of the thermotolerant bacteria against turfgrass pathogens was evaluated based on the size of the zone of inhibition after the confronting culture.
In addition, the experiment was performed using five petri dishes in each section and indicated by an average value of three repeated experiments. Table 3 shows the results.

[0053]

[Table 3]

As shown in Table 3, eight strains of thermostable bacteria obtained by screening from sewage sludge compost showed an antagonistic effect against the five turfgrass pathogens tested. In particular, two strains, AS-2-1 (Bacillus subtilus; Bacillus subtilus) and AS-2-2 (Bacillus firmus; Bacillus firmus), formed the largest zones of inhibition.

(Example 3) Production of turfgrass pathogen control agent-1 Two heat-resistant Bacillus strains which formed the largest zone of inhibition in Example 2 were each incubated at 30 ° C in a liquid nutrient medium.
For 7 days to form spores. This spore-forming culture was used alone or in an equal amount to form a mixture. 10 to 15 parts by weight of this culture was mixed with 90 to 85 parts by weight of diatomaceous earth (manufactured by Wako Pure Chemical Industries, Ltd.) and air-dried. After air-drying, the mixture was pulverized and mixed in a pot mill until uniform, whereby a turfgrass pathogen-controlling agent having a heat-resistant Bacillus bacterium CFU (colory-forming unit) of 2 to 8 × 10 ⁸ per g of diatomaceous earth was obtained.

(Example 4) Control agent for turfgrass pathogen-2 The two heat-resistant Bacillus strains which formed the largest zone of inhibition in Example 2 were each treated with 30 strains in a liquid nutrient medium.
The culture was obtained by shaking culture at 3 ° C. for 3 days. Each of the obtained cultures was used alone or mixed in an equal amount to obtain a mixture. This liquid mixture is packed in a mayonnaise bottle and kept at 121 ° C for 20 minutes.
Then, 10 to 20 parts by weight was inoculated with respect to 100 parts by weight of sterilized rice bran, and cultured at 55 ° C. for further 2 days to form spores. The solid culture was air-dried and pulverized with a pot mill to obtain a turfgrass pathogen-controlling agent having a CFU of 1 to 5 × 10 ⁹ per 1 g of rice bran.

Example 5 Production of Organic Fertilizer 11 parts by weight of urea, 30 parts by weight of lime superphosphate, 9 parts by weight of potassium chloride, and 50 parts by weight of sewage sludge were mixed well by a mixer. This mixture was pulverized, granulated, dried and sieved according to a known method for producing an organic fertilizer. In this way, each component of nitrogen (N): phosphorus (P): potassium (K) is added to the organic fertilizer at a weight ratio (w / w) of 5:
An organic fertilizer containing 5: 5 was obtained.

Example 6 Production of Organic Fertilizer Having a Coating Layer The same two heat-resistant Bacillus strains used in Example 5 were cultured in a nutrient liquid medium at 30 ° C. with shaking for 7 days. Or, after shaking culture at 30 ° C. for 3 days, further culturing at 60 ° C. for 2 days to form spores,
A spore-forming culture was obtained.

This culture was used alone or in an equal amount to form a mixed culture. To 1,000 parts by weight of this culture, 1 part by weight of trangant rubber was added and dissolved to prepare a coating solution. The whole amount of the coating solution was spray-coated on 10,000 parts by weight of the organic fertilizer obtained in Example 5 using an IK sprayer (manufactured by Environmental Equipment Company). As described above, CFU of heat-resistant bacillus is increased to 1 per gram of organic fertilizer.
An organic fertilizer having ２2 × 10 ⁸ of this coating layer was obtained.

(Example 7) Examination of the control effect of the turfgrass pathogen control agent of Example 3 To examine the control effect of the turfgrass pathogen control agent obtained in Example 3 on turfgrass pathogens, turfgrass tissues were used. The experiment was conducted as follows. That is, turfgrass (bent grass) is cultivated and collected in a garden at home, the tissue tissue including the leaves and stems of the turfgrass is dried, and the turfgrass is cut to a length of 5 to 10 mm with scissors to obtain an inner diameter of 8.5 cm. 3 per petri dish
g. Then, 5 ml of water was added to these petri dishes, and sterilized in an autoclave at 121 ° C. for 20 minutes.

After cooling the autoclaved petri dish, the turfgrass pathogen controlling agent obtained in Example 3 was washed with sterilized water for 200 hours.
It was diluted by a factor of 2 and 5.6 mL was sprayed on each of the above-mentioned petri dishes. This application rate corresponds to 1000 mL / m ² . Table 2
5 pathogens shown in Table 3 were inoculated into 20 mL of PDA to about 3 mm and pre-cultured. Each of the pre-cultured bacteria was punched out with a cork borer having an inner diameter of 6 mm, and inoculated into the center of the turfgrass tissue placed in each petri dish sprayed with the turfgrass pathogen controlling agent of the present invention. Each petri dish was cultured for 10 days at the optimal temperature for growth of the pathogenic bacteria, and the control effect was examined by measuring the elongation of the hyphae of the pathogenic bacteria.

As a positive control, capstan wettable powder (manufactured by Nippon Agrochemical Co., Ltd.) as a conventional pesticide was diluted 500-fold with sterile water, and sprayed onto the above petri dish containing turfgrass at 5.6 mL / dish. As a negative control, a petri dish containing only the above turfgrass was used. The experiment was repeated three times using five petri dishes in each section, and the average value was calculated and shown in Table 4.

[0063]

[Table 4]

As shown in Table 4, the turfgrass pathogen-controlling agent containing the heat-resistant bacillus spore-forming culture obtained in the present invention as an active ingredient remarkably suppressed hyphal elongation of the turfgrass pathogens tested. The spore-forming culture of the thermostable bacteria Bacillus subtilis and Bacillus famas obtained in Example 2 showed almost the same effect when used alone. In addition, when these cultures were mixed at a ratio of 1: 1, a higher inhibitory effect was obtained than when Bacillus subtilis was used alone. The turfgrass pathogen control agent of the present invention,
It showed the same level of inhibitory effect as Captan wettable powder.

Example 8 Examination of the Control Effect of the Control Agent for Turf Grass Pathogen in Example 6 The lawn pathogen of organic fertilizer having a coating layer containing the spore-forming culture of the heat-resistant bacillus of the present invention obtained in Example 6 The effect of controlling against turfgrass was examined using turfgrass tissue. Large patch disease and brown patch disease were used as turfgrass pathogens. The turfgrass tissue was placed in a petri dish having an inner diameter of 8.5 cm and sterilized in the same manner as in Example 7.

After cooling the autoclaved petri dish, the three kinds of organic fertilizers obtained in Example 6 were evenly and uniformly applied so as to obtain 0.28 g and 0.56 g per sterilized petri dish. This amount corresponds to an application rate of 50 g and 100 g per m ² . Thereafter, 2.8 mL and 5.6 mL of sterile water were sprayed on each petri dish. These application rates correspond to application rates of 500 mL and 1000 mL per m ² .

Then, a 6.0 mm disk prepared by culturing the test pathogen in the same manner as in Example 7 was inoculated on the turfgrass tissue placed at the center of the petri dish. These petri dishes were cultured in a 25 ° C. incubator, and the elongation of hyphae on day 8 was measured to examine the inhibitory effect. As a positive control organic fertilizer, commercially available sewage sludge-derived compost (trade name: Higashi Organic, Higashi Sangyo Co., Ltd.) was used. In Table 5, "No bacteria" was described.

As a negative control, a petri dish containing only the turfgrass treated as described above was used. In Table 5, "No application" was indicated. The experiment was repeated for three days using three petri dishes in each section, and the average value was obtained.

[0069]

[Table 5]

As shown in Table 5, the organic fertilizer having the coating layer containing the spore-forming culture of the thermostable Bacillus obtained in the present invention was applied at any of 50 g / m ² and 100 g / m ^2. , The hyphal elongation of large patch disease and brown patch disease was significantly suppressed. Incidentally, sewage sludge compost, although the application rate of 50 g / m ² did not inhibit the elongation of hyphae of pathogens, in the case of using 100 g / m ^2, the tendency to delay the extension of hyphae of pathogens was observed .

(Example 9) Examination of the control effect of the turfgrass pathogen control agent of Example 4 Using the turfgrass pathogen control agent containing the spore-forming culture of the heat-resistant bacillus obtained in Example 4 as an active ingredient, An experiment was conducted on the control effect of bentgrass on brown patch disease caused by nursery. The experimental scale was 1 m ² in each section. The application rate was measured by weighing 10 g of the turfgrass pathogen controlling agent of the present invention, adding the mixture to 1000 mL of water, stirring well, and spraying each section with Ryoden.

As a positive control, Glasten wettable powder (manufactured by Nippon Agrochemicals Co., Ltd.), which is a conventionally used pesticide, was diluted 500-fold with 1 L of water, and 1 m ² was added to each plot to obtain the turfgrass pathogen of the present invention. It was sprayed in the same manner as the control agent. Evaluation of the control effect was performed as follows. Each test plot was photographed in advance, and this photograph was scanned with a scanner, and the lesion area was read. Two weeks after the control agent of the present invention and a pesticide (glasten wettable powder) as a control were sprayed on each test plot, two weeks later, each test plot was again photographed. The photograph was scanned and the lesion area was read. The lesion area before spraying was compared with that after spraying, and the reduction rate of the lesion area was determined. The control value was calculated as follows using the reduction rate of the lesion area.

Control value (%) = (1-lesion remaining area ratio in plots using the control agent or pesticide of the present invention / lesion remaining area rate in non-application plot) × 100 The results are shown in Table 6.

[0074]

[Table 6]

As shown in Table 6, the agent for controlling turfgrass pathogens of the present invention containing a heat-resistant bacillus spore-forming culture as an active ingredient showed a remarkable control effect against brown patch disease. The control rate was almost comparable to that of Glasten wettable powder.

Example 10 Examination of the Control Effect of the Control Agent for Turf Grass Pathogen in Example 6-2 Rhizoctonia spring of an organic fertilizer having a coating layer containing the spore-forming culture of the heat-resistant bacillus obtained in Example 6 The control effect on baldness was tested. Experiment scale is 3 for each section
The size was 24 m ² of mx 8 m, and it was always performed on the Korai fairway (Koroshiba fairway) of a golf course where rhizoctonia spring baldness occurs. The amount used was 100 g of the organic fertilizer of the present invention per 1 m ² . As a control, a commercially available sewage sludge compost (East Organic) was used, and the amount used was similarly 100 g per 1 m ² .

The application of these fertilizers was carried out in early April, and the occurrence of the disease was investigated in late April. Take a picture of each test plot (test plot) on which fertilizer was applied on the day of the disease outbreak survey, scan it with a computer, process it with a computer,
The area of the lesion generated in the test area was read. The control effect was evaluated based on the control value, and the control value was calculated by the following formula.

The control value (%) = (1−the area ratio of lesions in the area where the organic fertilizer of the present invention was applied / the area ratio of lesions in the area where the organic fertilizer was not applied) × 100 The results are shown in Table 7.

[0079]

[Table 7]

As shown in Table 7, the organic fertilizer having the coating layer containing the spore-forming culture of the thermostable Bacillus obtained in the present invention showed a high control effect against Rhizoctonia scab.

(Example 11) Examination of the effect of maintaining the fertilizer effect of the organic fertilizer of Example 5 Using the organic fertilizer of the present invention obtained in Example 5, the effect of maintaining the fertilizer effect on turfgrass was examined. The fertility test was performed in a nursery of bentgrass. As the control fertilizer, green phosca (N-P-K ratio = 10-10-10, manufactured by Chisso Asahi Fertilizer) often used in golf courses was used. The experimental scale was two sections of 1 m ² × 1 m 1 m ² .

[0082] The amount, in order to equalize the amount of components N-P-K, the manure of the present invention 100 g / m ^2, a green host Ska was 50 g / m ^2. The fertilizer-retaining effect was measured using a whole cup (4.25
Two samples were taken out using an inch cup, and the number of buds, above-ground live weight and dry weight (all (mg / 10)) and root fresh weight and dry weight (all (mg / 10)) were examined. . The average values are shown in Table 8.

[0083]

[Table 8]

From the results shown in Table 8, when the organic fertilizer of the present invention was used, the number of buds, the live weight and the dry weight of the above-ground part and the rhizome part were higher than those in the untreated plot where no fertilizer was used. In both cases, there was a significant increase. In addition, compared to green fosca, a chemical fertilizer commonly used in golf courses, the number of buds and the fresh and dry weight of the rhizome greatly increased, indicating that turfgrass can be grown healthy. became. Therefore, it was shown that the organic fertilizer of the present invention is far superior to the conventional fertilizer in healthy growing turfgrass.

On the other hand, when the organic fertilizer of the present invention is used, the fresh weight of the above-ground part is smaller than that of green fosca which is a chemical fertilizer. This means that when using chemical fertilizers, the growth of the above-ground part is faster than that of the rhizome, but when using the organic fertilizer of the present invention, the growth of the rhizome is faster than the growth of the above-ground part. Is shown. Therefore, when the organic fertilizer of the present invention is used, turfgrass is firmly rooted,
It is suggested that the above-ground mother is not as prolonged as when using chemical fertilizers, which can reduce the number of turf cuts. When the number of times of cutting is reduced, not only can the lawn grass management of the golf course be labor-saving, but also the number of times that the stems and leaves of the lawn grass are damaged by the cutting can be reduced, so that the chance of contact with pathogenic bacteria can be reduced.

(Example 11) Changes over time in the number of spore-forming bacteria in the turfgrass pathogen control agent and the organic fertilizer of the present invention The turfgrass pathogen control agent obtained in Examples 3 and 4, and the organic substance obtained in Example 6 How the heat-resistant Bacillus of the present invention contained in the fertilizer changes with time was examined as follows, and the stability of these bacteria was examined. 1 g of the turfgrass pathogen control agent obtained in Examples 3 and 4 was weighed and diluted with sterilized water to an appropriate magnification. This diluted solution was spread on a nutrient agar medium previously poured into a petri dish, cultured at 30 ° C. for 72 to 120 hours, and cfu was calculated from the number of colonies that appeared on the agar.

10 g of the organic fertilizer obtained in Example 6 was weighed, placed in 90 mL of sterilized water, and treated with ultrasonic waves for 15 minutes. This solution was diluted with sterile water to an appropriate magnification, applied on a nutrient agar medium in the same manner as in the method described above, and heated at 30 ° C for 72 to
After culturing for 120 hours, cfu was calculated from the number of colonies that appeared on the agar. Table 9 shows the results.

[0088]

[Table 9]

As shown in Table 9, even after one year from the preparation of the turfgrass pathogen-controlling agent of the present invention, the number of heat-resistant Bacillus contained in the controlling agent of the present invention did not significantly decrease. I was not able to admit. The number of Bacillus bacteria contained in the organic fertilizer having the coating layer was more than 100% even after one year.

That is, it has been clarified that the heat-resistant bacillus of the present invention still maintains stability even after one year from the prescription. In addition, since these Bacillus can withstand high temperatures, it can be used as a microbial material for making a composition that is often heated during long-term storage and transportation, such as the above-mentioned control agents and organic fertilizers. it is obvious.

[0091]

According to the present invention, there is provided a pesticide for turfgrass pathogenic bacteria which is a pesticide which has a high effect on pathogenic bacteria with a small amount of use and does not cause environmental pollution or water pollution. Further, there is provided an organic fertilizer which can be used on both warm-season turf and cold-season turf cultivated in a vast golf course.

The controlling agent of the present invention can be used for golf course greens,
It can be used anywhere, including tea grounds, fairways, roughs and nurseries. In addition, it is possible to suppress the occurrence of various turfgrass diseases such as brown patch disease, yellow patch disease, and red blight disease, and to prevent the spread of the disease.

Further, the organic fertilizer of the present invention not only can grow turfgrass soundly, but also can maintain the green color of turf for a long time. Therefore, the amount of pesticides used in the golf course can be reduced, and the amount of fertilizer used can be reduced. When the organic fertilizer of the present invention is applied, the growth of the rhizome is faster than the growth of the above-ground part, so that the number of times of cutting can be reduced, which contributes to labor saving of golf course management. According to the present invention, a method for producing such a pesticide and an organic fertilizer is also provided.

Claims (14)

[Claims] A heat-resistant Bacillus subtilis or Bacillus famas which suppresses the growth of turfgrass pathogens obtained by autoclaving sewage sludge. 2. The heat-resistant Bacillus subtilis or Bacillus famas according to claim 1, wherein the autoclave treatment is a sterilization treatment at 121 ° C. for 20 minutes. 3. A turfgrass pathogen-controlling agent comprising the spore-forming culture of Bacillus subtilis and / or Bacillus famas according to claim 1 or 2 alone and / or as a mixture. 4. The method according to claim 1, wherein the spore-forming culture is cultured at 25 ° C. to 30 ° C. for 2 hours.
A turfgrass pathogen-controlling agent, which is obtained by culturing for 3 to 3 days and then at 55 ° C to 65 ° C for 2 to 3 days. 5. A step of autoclaving the sewage sludge compost, a step of screening heat-resistant Bacillus subtilis and / or Bacillus famas from the autoclaved sewage sludge compost, and a spore-forming culture by spore-forming the fungus. Obtaining the spore-forming culture alone or in a mixture at a ratio of 7: 3 to 1: 1 to form a mixture, and allowing the mixture to adsorb to the base at 2 to 8 × 10 ⁸ cfu / g; And a step of pulverizing turfgrass. 6. An organic fertilizer containing nitrogen (N): phosphoric acid (P): potassium (K), 4 to 8% by weight each, and sewage sludge. 7. The organic fertilizer according to claim 6, further comprising a coating layer containing the spore-forming culture of Bacillus subtilis and / or Bacillus famas according to claim 1 or 2. 8. The method of claim 1, wherein the coating is
The organic fertilizer according to claim 6, wherein the spore-forming culture of the bacterium according to (1) or (2) comprises a viscous polysaccharide alone or a mixture thereof. 9. The organic fertilizer according to claim 8, wherein said viscous polysaccharide is selected from the group consisting of tragacanth gum, galacturonic acid, fucose, and gum arabic. 10. The coating layer according to claim 6, wherein the spore-forming culture and the viscous polysaccharide are mixed at a ratio of 500: 1 to 1000: 1. Organic fertilizer. 11. The process according to claim 1, wherein nitrogen (N), phosphoric acid (P), and potassium (K) are added to the sewage sludge to a concentration of 4 to 8% by weight to form a mixture. Preparing a coating solution comprising a spore-forming culture of Bacillus subtilis and / or Bacillus famas and a viscous polysaccharide; and coating the mixture with the coating solution. Organic fertilizer production method. 12. The method for producing an organic fertilizer according to claim 10, wherein the spore-forming culture is a culture of the fungus according to claim 1 alone or as a mixture. 13. The method for producing an organic fertilizer according to claim 11, wherein the viscous polysaccharide is selected from the group consisting of tragacanth gum, galacturonic acid, fucose, and gum arabic. 14. The organic fertilizer according to claim 11, wherein the mixture ratio of the spore-forming culture and the viscous polysaccharide is from 500: 1 to 1000: 1. Manufacturing method.