JPH08175921A - Agricultural and horticultural germicidal composition - Google Patents

Abstract

PURPOSE: To obtain an agricultural and horticultural germicidal composition, excellent in disease injury controlling actions and good in colonization properties and persistence stability by utilizing a bacterium of the genus Bacillus. CONSTITUTION: This agricultural and horticultural germicidal composition contains spores of a bacterium belonging to the genus Bacillus, preferably a bacterium Bacillus subtilis FERM P-14647 strain or Bacillus subtilis FERM P-14646 strain and a spore germination promoter, preferably a nutrient substrate, a chelating compound or an inorganic salt. The spores are obtained by carrying out the solid culture of the bacterium belonging to the genus Bacillus or the liquid culture thereof under aerobic conditions, centrifuging the resultant cultured product and removing the culture supernatant. The content of the spores in the germicidal composition is preferably 0.001-99.9 pts.wt. and that of the spore germination promoter is preferably 0.1-99.999 pts.wt. based on the total amount of the composition. Methods for coating seeds therewith, treating a single flower, treating leaves or stems, coating a wound site or a pruned part of the cultivated plants, irrigating soil or mixing soil therewith, etc., are cited as the method for applying the composition.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an agricultural / horticultural fungicide composition, and more particularly to an agricultural / horticultural fungicide composition containing a spore of a bacterium belonging to the genus Bacillus and a spore germination promoter and having an excellent disease control effect. Regarding things.

[0002]

2. Description of the Related Art In recent years, as a method of protecting agricultural and horticultural plants from various diseases, in consideration of safety and sustainability of effects, the occurrence of diseases is prevented by using microorganisms that compete with pathogenic bacteria that cause various diseases. The method is widely used.

Microorganisms that have been used for controlling the diseases of such agricultural and horticultural plants include bacteria belonging to the genera Trichoderma, Gliocladium, Bacillus, and the like, and farms containing these microorganisms so far. Many horticultural germicide compositions have also been researched and developed.

Regarding the bacteria belonging to the genus Bacillus among them, for example, in JP-A-63-273470, Bacillus subtilis JB3 strain (NCIB12375), Bacillus subtilis JB
Antibacterial substances obtained from the 3.6 strain (NCIB12376), Bacillus subtilis R1 strain (NCIB12616) or mutants thereof suppress plant diseases, microbial infections of animals and humans, and further prevent general microbial contamination. As a control, attempts have been made to control diseases of various agricultural and horticultural plants using cultures of the above strains.

Further, in Japanese Patent Laid-Open No. 22222/1990,
Bacillus subtilis JB above
Antibiotics derived from 3 strains (NCIB12375), Bacillus subtilis JB3.6 strain (NCIB12376), Bacillus subtilis R1 strain (NCIB12616) or mutants thereof are isolated, and these are used as a disease of various agricultural and horticultural plants. Attempts have been made to use it for control.

[0006] However, none of the methods of controlling diseases of agricultural and horticultural plants using such bacteria belonging to the genus Bacillus is not always sufficient in terms of sustainability, fixability and stability.

[0007] It is known that such bacteria belonging to the genus Bacillus form spores, and spores are often isolated from cultures. No examples have been reported so far, which have been prepared in a large amount, or cases where spores were combined with a spore germination promoter to try to show the effect of controlling disease on agricultural and horticultural plants.

[0008]

The present invention has been made from the above point of view, and in an agricultural and horticultural fungicide composition utilizing a bacterium belonging to the genus Bacillus, excellent in disease control action, and further, fixability, It is an object of the present invention to provide a fungicide composition for agricultural and horticultural use with good long-term stability.

[0009]

Means for Solving the Problems As a result of intensive research to solve the above problems, the present inventor took out spores from a culture of a bacterium belonging to the genus Bacillus, and used them together with a spore germination promoter for agriculture and horticulture. It was found that an agricultural and horticultural fungicide composition having an excellent disease control action, a good fixability and a long-term stability can be obtained by adding it to the fungicide composition for agricultural use, and completed the present invention.

That is, the present invention is an agricultural and horticultural fungicide composition containing spores of a bacterium belonging to the genus Bacillus and a spore germination promoter. Hereinafter, the present invention will be described in detail.

<1> Bacterial spores belonging to the genus Bacillus The agricultural / horticultural fungicide composition of the present invention contains spores obtained from a culture of bacteria belonging to the genus Bacillus as an active ingredient.

The bacterium belonging to the genus Bacillus used in the present invention is not particularly limited as long as it is a bacterium belonging to the genus Bacillus, but preferably a bacterium belonging to the genus Bacillus which competes with a plant pathogenic bacterium, among which more preferred Include Bacillus subtilis, and among them, Bacillus subtilis FERM
P-14647 strain, Bacillus subtilis FERM
P-14646 strain is preferable.

Bacillus subtilis FERM P-14
647 strain and Bacillus subtilis FERM P-1
The strain 4646 has been deposited with the Institute of Biotechnology, Industrial Technology Institute (FERM) on November 17, 1994. Also, Bacillus subtilis FERM P-14
Strain 647 is NCIB12376 strain, Bacillus subtilis FERM P-14646 strain is NCIB1 strain
As strain 2616, Scotland, AB98DG,
Aberdeen, Abbey Ro
ad) 135, P.I. O. Box 31 National Collections of Industrial and Marine Bacteria
and Marine Bacteria Ltd.) (NCIB), 1 at Torry Research Station
December 22, 986 (NCIB12376), 19
Deposited on Dec. 24, 1987 (NCIB12616), the nature of these strains is described in European Patent Application Publication No. 2
No. 76132.

The spores used in the present invention are obtained from a culture of the bacterium belonging to the genus Bacillus. Cultivation of bacteria belonging to the genus Bacillus can be carried out in accordance with ordinary culturing methods of bacteria belonging to the genus Bacillus, for example, liquid culture such as reciprocal shaking culture, jar-famenter culture, culture tank culture, and solid culture. .

The medium used for the culture may be any medium as long as it can easily form spores efficiently. As carbon sources, sugars such as glucose, starch, dextrin, sucrose and molasses, organic acids such as citric acid and malic acid, Alcohols such as glycerin are used as nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium salts such as ammonium nitrate and nitrates and yeast nitrogen, corn steep leaker, meat extract, wheat germ, polypeptone, soybean flour and other organic nitrogen sources. As the inorganic salt, salts of phosphoric acid, potassium, calcium, manganese, magnesium, iron and the like, for example, potassium chloride, calcium chloride, manganese sulfate, ferrous sulfate and the like can be added. Further, various additives such as an antifoaming agent can be used if necessary.

The culture conditions are not particularly limited, but the culture is preferably solid culture or liquid culture performed under aerobic conditions such as aeration and shaking culture, and the temperature is preferably 10 to 50. C, more preferably 15-40
C. and pH are preferably in the range of 4 to 9, more preferably 6 to 8.

The method for separating spores from the culture belonging to the genus Bacillus obtained as described above can be carried out by a method such as membrane separation, centrifugation or filtration separation.
The resulting spore fraction may be used as it is in the agricultural and horticultural fungicide composition described below in a state of containing a certain amount of water, and if necessary, a drying method such as freeze drying, ventilation drying, and spray drying. It can also be used as a dried product in an agricultural / horticultural germicide composition.

A method for producing spores of a bacterium belonging to the genus Bacillus used in the present invention is described in, for example, Bacillus subtilis F.
The case of using the ERM P-14647 strain will be described. Bacillus subtilis FERM P-14647
The slope culture of the strain is inoculated into a Sakaguchi flask containing broth medium (meat extract 1%, peptone 1%, NaCl 0.5%), and then cultured at 30 ° C. for 1 day on a rotary shaker. The obtained culture was used as a medium (glucose 2%, polypeptone 1
%, Yeast extract 0.2%, KH ₂ PO ₄ 0.1%) was inoculated into the fermenter and cultured under aerobic conditions at 30 ° C. for 72 hours to obtain a culture solution. This culture solution is centrifuged to separate it into a culture supernatant and a bacterial cell precipitate, and after removing the culture supernatant, the bacterial cell precipitate is washed with water to obtain wet bacterial cells (spore fraction). Further, this spore fraction (wet cell) is frozen at −80 ° C., dried under reduced pressure and pulverized to give a spore fraction as a dried product. Bacillus subtilis FE obtained in this way
The spore fraction of the RM P-14647 strain culture contains the spores of the strain in an amount of about 50% to 100% by dry weight.

<2> Spore Germination Promoter In the agricultural / horticultural fungicide composition of the present invention, a spore germination promoter is added together with the spores of the bacteria belonging to the genus Bacillus.

The spore germination promoter used in the present invention includes:
There is no particular limitation as long as it is known as a component that promotes germination of spores of bacteria belonging to the genus Bacillus, and examples of the nutrient substrate include glycerol, L-arabinose, ribose, and D-xylose, which are carbon sources. , Galactose, D-glucose, D-fructose, D-
Mannose, inositol, mannitol, D-sorbitol, α-methyl-D-glucoside, N-acetylglucosamine, amygdalin, arbutin, salicin, cellobiose, maltose, lactose, melibiose,
Sucrose, trehalose, inulin, D-raffinose, starch, dextrin, glycogen, β-gentibose, D-tulanose, α-starch, soluble starch, licorice sweetener, erythritol, chitin, chitosan, cellulose, glycyrrhizin, coupling sugar, Maltitol, maple sugar, cationized starch, invert sugar, honey, starch syrup, oligosaccharides and polysaccharides composed of one or more of the above sugars, citric acid, etc., various amino acids as nitrogen sources, ammonium sulfate , Ammonium phosphate, ammonium nitrate, ammonium chloride, urea, soybean powder, rice straw, and other components such as yeast extract and peptone.

As a spore germination promoter, in addition to the above nutrient substrates, inorganic salts such as potassium phosphate, iron phosphate, lime doubly superphosphate, ammonium polyphosphate, condensed phosphate (sodium polyphosphate, polyphosphate) Acid potassium, etc.), acidic phosphates, water-soluble inorganic phosphates such as sodium hexametaphosphate, potassium salts such as potassium chloride and potassium nitrate, salts of trace elements such as copper, iron, zinc, calcium, magnesium, manganese and molybdenum. Etc. can be mentioned. Although these inorganic salts can promote germination of spores when used alone, they are preferably used together with sugars, and more preferably together with glucose among sugars.

EDTA, sodium hydrogen carbonate, sodium thioglycolate, glycylglycine, 8-hydroxyquinoline, oxalic acid, tartaric acid, citric acid, sorbitol, phosphoric acid, sodium metaphosphate, pyrophosphate, sodium tripolyphosphate, tripolyline A chelate compound such as potassium acid salt or potassium metaphosphate can also be added to the agricultural or horticultural fungicide composition of the present invention as a spore germination promoter.

<3> Agro-horticultural fungicide composition The agricultural-horticultural fungicide composition of the present invention contains one or more spores and spore germination promoters of the bacteria belonging to the genus Bacillus, respectively. is there.

When the spores of the bacterium belonging to the genus Bacillus are added to the agricultural and horticultural fungicide composition of the present invention, the weight of the contained spores should be 0.001 to 99.9 parts of the total amount of the composition. It is preferable to mix them. Spore content is 0.001
If it is less than 1 part by weight, the disease controlling effect may not be sufficient, and if it exceeds 99.9 parts by weight, an effective amount of the spore germination accelerator cannot be formed in the agricultural and horticultural bactericidal composition, and thus the disease controlling is also impossible. The effect may not be sufficient.

The amount of the spore germination promoter is preferably 0.1 to 99.999 parts by weight with respect to the total amount of the composition. When the content of the spore germination accelerator is less than 0.1 part by weight, an excellent disease control effect may not be obtained,
If the amount exceeds 99.999 parts by weight, a sufficient amount of spores to exert a disease controlling effect cannot be formed in the agricultural and horticultural germicidal composition, and the excellent disease controlling effect by the spores cannot be obtained. is there.

The fungicide composition for agricultural and horticultural use of the present invention comprises a spore of a bacterium belonging to the genus Bacillus and a spore germination promoter, if desired, together with various optional components, and a powder or granules according to a conventional method for producing a microbial preparation. It is formulated into an agent, a wettable powder, an emulsion, a liquid agent, a flowable agent, a coating agent and the like.

The above-mentioned optional components include solid carriers,
Carion clay, pyrophyllite clay, bentonite, montmorillonite, diatomaceous earth, synthetic hydrous silicon oxide,
Fine mineral powder such as acid clay, talc, clay, ceramics, quartz, sericite, vermiculite, perlite, Otani stone, anthra stone, limestone, coal ash, zeolite, etc.
Rice husk, bran, crab husk, shrimp husk, fine krill powder, rice bran, wheat flour, corn cob, peanut husk, bone meal, fish meal, meal, sawdust, wood meal, charcoal, kun charcoal, bark charcoal, husk husk charcoal, grass Examples thereof include charcoal, peat moss, attapulgite, dried feces, activated carbon, and fine powder of organic matter such as oil cake. Examples of the liquid carrier include water, vegetable oil, liquid animal oil, synthetic water-soluble polymer (polyvinyl alcohol, polyvinylpyrrolidone, polyacrylic acid, etc.) and the like. Further, as an auxiliary agent, if necessary, a soluble extender,
Casein, gelatin, gum arabic, alginic acid, synthetic polymers (polyvinyl alcohol, polyacrylic acid, etc.), bentonite and other fixing agents and dispersants, and other components such as propylene glycol, ethylene glycol and other antifreezing agents, xanthan gum, etc. Examples thereof include thickeners such as natural polysaccharides and polyacrylic acids.

The plant pathogens to which the fungicide composition for agricultural and horticultural use of the present invention thus obtained is applied, those pathogens belonging to molds, for example, Rhizoctonia solani, a rice wilt disease fungus. ), The blast fungus Pyricularia oryzae, and the powdery mildew fungus Erysiphe graminis of barley.
graminis), wheat wilt fungus Geumanomyces graminis, pea brown spot fungus Ascochyta pisi, broad bean red spot fungus Botrytis fabae (Botrytis faba)
e),

Vegetables such as tomato, eggplant, strawberry,
Botrytis cinerea, a fungus that causes gray mold such as cucumber, lettuce, and green beans, and Alternaria brass
icicola), tomato leaf mold fungus Cladosporium fulvum, phytophthora infestans Phytophthora infestans, Fusarium oxysporu
m), powdery mildew fungus of cucumber Sphaerotheca fuliginea, downy mildew fungus Pseudoperonospora cubensi
s), wilt fungus Pythium sp., rust rust fungus Puccinia allii, Sclerotinia allii
Chinese cabbage leaf spot fungus Alternaria brassicae (Altern
aria brassicae), carrot black leaf blight fungus Alternaria dauci, spinach wilt fungus Rhizoctonia solani, lettuce sclerotinia sclerothiolam (Sclero)
tinia sclerotiorum), Verticillium dahliae, a half-body wilt fungus of eggplant, and powdery mildew fungus Sphaerotheca of strawberry.
humuli), anthracnose fungus
etotrichum fragariae),

[0030] Flowers such as cyclamen, chrysanthemum, rose, and statice Botrytis cinerea, powdery mildew fungus Sphaerotheca pannosa, white rust fungus Puccinia holiana (Puccinia) horiana),

[0031] Pseudomonas aeruginosa (Rosellinia necatrix) on fruit trees, Penicillium italicu (Penicillium italicu) on citrus
m), Black spot bacterium Diaporthe citr
i), Gymnosporangium asiaticum, a red scab of pear, Venturia inaequali, a black scab of apple, Venturia inaequali
s), Peach scab, Monilinia fructicola (Mon
ilinia fructicola),

[Large patch disease fungus Rhizoctonia solani (Rhizoctonia solani) of the lawn, leaf blight fungus curvularia
sp (Curvularia sp), Helmint sporium sp
(Helminthosporium sp), rust fungus Puccinia zoysiae, dollar spot fungus Sclerotinia homeocarpa (Sclerotinia homoeocarp)
a), spring blight fungus Fusarium, Rhizoctonia, Pythium, snow rot Typhula incarnata, brown patch fungus Rhizoctonia sola
ni) and the like.

<4> Method for Applying Agricultural and Horticultural Fungicide Composition of the Present Invention The agricultural and horticultural fungicide composition of the present invention is applied to cultivated plants for the purpose of controlling various diseases of the various cultivated plants as described above. However, the method is appropriately selected depending on the usage form such as dosage form, crop and disease, and for example, above-ground liquid spray, above-ground solid spray, aerial liquid spray, aerial solid spray, water surface application, facility application, soil mixing. Examples thereof include application, soil irrigation application, surface treatment (seed dressing, application treatment, etc.), nursery box application method, single flower treatment, and plant base treatment. Further, among these application methods, preferably, the seeds of cultivated plants are coated with various dosage forms of agricultural and horticultural fungicide compositions, the flowers of cultivated plants are subjected to single flower treatment, the foliage of cultivated plants is treated, Examples of the method include applying treatment to the wound site and pruning part of the cultivated plant, irrigating the soil, and mixing the soil.

In addition, when applying the agricultural and horticultural fungicide composition to cultivated plants, insecticides, nematicides, acaricides, herbicides, fungicides, plant growth regulators, fertilizers, soil improving materials (peat) , Humic acid materials, polyvinyl alcohol materials, etc.) may be mixed and applied, or they may be alternately applied without mixing or simultaneously applied.

The application rate of the fungicide composition for agricultural and horticultural use of the present invention varies depending on the type of disease, the type of plant to be applied, the dosage form of the fungicide composition, etc., but cannot be specified unconditionally. When the horticultural germicide composition is sprayed on the ground, the spore concentration for application is usually about 10 ⁶ CFU (colony forming unit) / mL to ^{10 10} CFU / mL, preferably about 10 ⁷ CFU / mL. -10 ⁹ CFU / mL, application rate is 0.5-50 L / a. In addition, granules, powders, etc. can be applied as they are without any dilution, and when sprayed on the ground, the spore application rate is about 10 ¹² to 10 ¹⁴ CFU / a. Preferably.

[0036]

Embodiments of the present invention will be described below. First, an example of the production of spores of a bacterium belonging to the genus Bacillus that is added to the agricultural / horticultural germicide composition of the present invention will be described.

[0037]

[Production Examples 1 and 2] Spore fraction of a bacterium belonging to the genus Bacillus (wet cell) One platinum loop of a slope culture of a preservation bacterium of Bacillus subtilis FERM P-14647 strain is 100 m per flask.
L broth medium (meat extract 1%, peptone 1%, Na
Sakaguchi flask containing 500% Cl (500 mL)
After inoculation, the cells were cultured for 1 day at 30 ° C. using a reciprocal shaker with an amplitude of 10 cm and a rotation speed of 120 rpm. 300 mL of the obtained culture was used as a medium (glucose 2%, polypeptone 1%, yeast extract 0.2%, KH ₂ PO ₄ 0.1%).
A 30 L fermenter containing L was inoculated and cultured under aerobic conditions at 30 ° C. for 72 hours to obtain a culture solution. Obtained about 1
Centrifuge 5 liters of culture broth according to the standard method (6000 rp
m, 20 minutes) to separate the culture supernatant and the bacterial cell precipitate.
After separating the culture supernatant as an antibiotic fraction (Comparative Example 1), the bacterial cell precipitate was washed with water to obtain a wet weight of about 780 g of wet bacterial cells (spore fraction) (Production Example 1). This spore fraction contains 100% by dry weight of spores of Bacillus subtilis FERM P-14647.

In the above, Bacillus subtilis FERM P
Instead of using the 14647 strain, the same operation was performed using the Bacillus subtilis FERM P-14646 strain, and a wet weight of about 750 containing 50% by dry weight of spores of the Bacillus subtilis FERM P-14646 strain was used.
A spore fraction of g was obtained (Production Example 2).

[0039]

[Comparative Example 2] Viable cell fraction of bacteria belonging to the genus Bacillus (wet cells) One platinum loop of a slope culture of a preservation bacterium of Bacillus subtilis FERM P-14647 strain was 100 m per flask.
L broth medium (meat extract 1%, peptone 1%, Na
Sakaguchi flask containing 500% Cl (500 mL)
After inoculation, the cells were cultured for 1 day at 30 ° C. using a reciprocal shaker with an amplitude of 10 cm and a rotation speed of 120 rpm. 300 mL of the obtained culture was used as a medium (glucose 2%, polypeptone 1%, yeast extract 0.2%, KH ₂ PO ₄ 0.1%).
A 30 L fermenter containing L was inoculated and cultured under aerobic conditions for 18 hours to obtain a culture solution. About 15 L of the obtained culture solution was centrifuged (6000 rpm, 20 minutes) according to a conventional method to separate into a culture supernatant and a bacterial cell precipitate. After removing the culture supernatant, the microbial cell precipitate was washed with water to obtain a wet microbial cell (viable microbial cell fraction) having a wet weight of about 600 g. This viable cell fraction contains 3% by dry weight of spores of Bacillus subtilis FERM P-14647 strain.

[0040]

[Production Examples 3 and 4] Powder spores Bacillus subtilis obtained in the above Production Example 1
About 600 g of the spore fraction of the FERM P-14647 strain was suspended in 3 L of water and then treated with a spray dryer (manufactured by Niro Japan) at a flow rate of 1.5 to 2 L / hr (inlet temperature 150 ° C., outlet temperature 100 ° C.). . The dried product obtained by the spray dryer was pulverized to obtain a powder spore having a dry weight of about 78 g (Production Example 3).

Similarly, about 78 g of Bacillus subtilis FERM was obtained from about 600 g of the spore fraction obtained in Production Example 2.
A powder spore of P-14646 strain was obtained (Production Example 4).

[0042]

Comparative Example 3 Viable Cell Suspension Bacillus subtilis Obtained in Comparative Example 2
About 100 g of the viable cell fraction of the FERM P-14647 strain was suspended in 10 L of water to obtain a viable cell suspension.

Next, examples of the fungicide composition for agricultural and horticultural use of the present invention containing the spore fraction of the bacterium belonging to the genus Bacillus and the spore germination promoter obtained in each of the above Production Examples will be described. In addition, all compounding amounts in the examples are parts by weight.

[0044]

Examples 1 to 6 Wettable powder The components shown in Table 1 were well mixed to obtain a wettable powder. Similarly, a wettable powder of Comparative Example containing spores of a bacterium belonging to the genus Bacillus but not containing a spore germination promoter was produced.

[0045]

[Table 1]

[0046]

Example 7 Wettable Powder The spore fraction obtained in the above Production Example 2 and the synthetic hydrous silicon oxide powder were mixed at a weight ratio of 2: 1 to adjust the water content, and the spore having a water content of 6% by weight. A mixture of the fraction and the synthetic hydrous silicon oxide powder was obtained. The respective components shown in Table 2 including this were thoroughly mixed to obtain a wettable powder. Similarly, a wettable powder of Comparative Example containing spores of a bacterium belonging to the genus Bacillus but not containing a spore germination promoter was produced.

[0047]

[Table 2]

[0048]

Example 8 Granules The ingredients shown in Table 3 were mixed well, and 10 parts of water was added to 90 parts of the obtained mixture and kneaded well, and then the diameter was 0.8 m.
It was granulated by an extrusion granulator equipped with a m screen and dried at room temperature. This was further sized to 300 to 1700 μm to obtain a granule. Further, in the same manner, a granule of Comparative Example containing spores of a bacterium belonging to the genus Bacillus but not containing a spore germination promoter was produced.

[0049]

[Table 3]

[0050]

Example 9 Dust Powder The ingredients shown in Table 4 were well mixed to obtain a dust powder. In the same manner, a comparative powder containing spores of a bacterium belonging to the genus Bacillus but not containing a spore germination promoter was produced.

[0051]

[Table 4]

<Evaluation of the agricultural and horticultural fungicide composition of the present invention>
The plant disease control effect of the agricultural and horticultural fungicide composition obtained in each of the above-mentioned examples together with the formulation of Comparative Example, turf, rose, lettuce,
The evaluation was performed using agricultural and horticultural plants such as tomato, green beans and eggplant.

(1) Shiba Rhizoctonia brown patch disease control
Removal test Culturing Rhizoctonia disease in a deep-bottomed petri dish (9 cm in diameter)
(PDA medium), sterilized soil is overlaid for about 8 minutes, and
The soil was prepared by culturing for 10 days. This contaminated soil surface
Of turfgrass (Bentgrass pencloth) after crushing
The pups were sown at a rate of 0.02 g per dish. Sowing
Immediately thereafter, a 200-fold dilution of the wettable powder obtained in Example 2
(1.0 x 10¹²CFU / L) 6.4 per dish
mL (1m ²(Equivalent to 1 L). This shah
Cultivated under 25 to 30 ° C and high humidity conditions, and after 15 days
The degree of disease was visually determined according to the following evaluation criteria.

+: Mild onset ++: Moderate onset +++: Severity onset

Further, the same test was conducted using a 200-fold diluted solution (1.0 × 10 ¹² CFU / L) of the wettable powder obtained in Examples 3 and 4. In addition, the same test was performed on the turfgrass that was not treated with the wettable powder obtained in each of the above-mentioned examples to investigate the degree of disease.

For comparison, 20 of the wettable powder of the above example was used.
20 of the wettable powder obtained in Comparative Example 4 was used instead of the 0-fold diluted solution.
The same test was conducted using a 0-fold diluted solution (1.0 × 10 ¹² CFU / L) to investigate the degree of disease onset. The results are shown in Table 5.

[0057]

[Table 5]

(2) Turf Rhizoctonia Brown Patch Disease Control Test A lawn rhizoctonia brown patch disease control effect was tested (variety: bentgrass pen cloth, test section: 10).
m ² / district, 2 repetitions). The attack was spontaneous. The powder agent obtained in Example 9 was added to river sand having a particle size of 0.1 to 1.0 mm so as to have a content of 10% by weight, and the mixture was uniformly mixed to obtain a soil for sand. 0.5m ^3/10 to ward
It was sprayed three times at an interval of 3 weeks from the end of May at a rate of 00 m ² . The diseased area was measured 3 weeks after the final application, and the diseased area ratio was obtained.

Further, the same test was carried out on the turfgrass which was not treated with the soil for soil containing the dust obtained in the above Example 9 to determine the diseased area ratio. The control value was calculated.

[0060]

[Equation 1] Control value (%) = (1- (the disease area ratio of the test example /
Control disease area ratio)) x 100

For comparison, the same test was carried out using the powder obtained in Comparative Example 7 instead of the powder of Example 9 above, the diseased area ratio was determined, and the control value was calculated. The results are shown in Table 6.

[0062]

[Table 6]

(3) Turbine dollar spot disease control test A control effect on the lawn dollar spot (scleroticinia disease) was tested (variety: bentgrass pen cloth, test section: 2 m ² / section, 3 repetitions). The attack was spontaneous. A 200-fold diluted liquid of the wettable powder obtained in Example 5 (1.
0 × 10 ¹² CFU / L) was soil-irrigated (1 L / m ² ) four times at 7-day intervals, and the number of lesions was examined 7 days after the final treatment.

Further, the same test was carried out on the turfgrass obtained in Example 5 which had not been treated with the wettable powder, and the number of lesions was investigated. Further, for comparison, instead of the 200-fold dilution of the wettable powder of Example 5 above, a 200-fold dilution of the wettable powder obtained in Comparative Example 4 (1.0 × 10 ¹² CFU /
L), commercially available bacitac wettable powder (main component; mepronil,
The same test was performed using Kumiai Chemical Co., Ltd. (2000 ppm) to investigate the number of lesions. The results are shown in Table 7.

[0065]

[Table 7]

(4) Turbine dollar spot disease control test The control effect on the lawn dollar spot (sclerotinia disease) was tested (variety: bentgrass pen cloth, test section: 10 m ² / section, no repetition). The attack was spontaneous. 30 g per 1 m ^{2 of} the granules obtained in Example 8
(6.0 × 10 ¹⁰ CFU / m ² ), sprayed 3 times at 7-day intervals, and the number of lesions was examined 7 days after the final spraying.

Further, the same test was carried out on the turfgrass obtained in Example 8 which had not been treated with the granules, and the number of lesions was investigated. Further, for comparison, Example 8 above
The same test was performed using the granules obtained in Comparative Example 6 (the granules having a spore content of 30 times that of Example 8 but containing no spore germination promoter) in place of the granules of The number of lesions was investigated. Table 8 shows the results.

[0068]

[Table 8]

(5) Test for controlling powdery mildew of roses A test for controlling powdery mildew of roses was conducted in a greenhouse (variety: Christian Dior, test section:
1 strain / group, 3 repetitions). The rose powdery mildew fungus was inoculated by arranging pots in which roses infected with the powdery mildew fungus were placed between the test plots. A 200-fold diluted solution of the wettable powder obtained in Example 5 (1.
0 × 10 ¹² CFU / L) was sprayed 3 times at intervals of 7 days (spray treatment (stem-and-leaf part spraying), 100 mL / strain) on the whole strain, and the diseased leaflet rate was examined 7 days after the final spraying. In addition, the same test was performed for roses that were not treated with the wettable powder obtained in Example 5 above to calculate the diseased leaflet rate, and using this as a control, a rose test was performed in the same manner as in the above-mentioned turfgrass test example. The control value of the example was calculated.

Further, for comparison, instead of the 200-fold dilution of the wettable powder of Example 5 above, a 200-fold dilution of the wettable powder obtained in Comparative Example 4 (1.0 × 10 ¹² CFU / L), a similar test was carried out using a commercially available suprol emulsion (main component: triphorin, manufactured by Takeda Pharmaceutical Co., Ltd., 1000 ppm) to investigate the diseased leaflet rate and calculate the control value. The results are shown in Table 9.

[0071]

[Table 9]

(6) Lettuce Sclerotial Disease Control Test The control effect against lettuce sclerotium disease was tested (variety: Cisco,
Test section: 40 strains / section, 3 repetitions). The inoculation of the lettuce sclerotium bacterium was carried out by arranging uniformly the lettuce sclerotium discs formed in a unglazed pot within the test plot. 250 L / 10 of a 500-fold diluted solution of the wettable powder obtained in Example 6 (3.0 × 10 ¹¹ CFU / L) was added to lettuce in the early stage of heading.
Sprayed 3 times at intervals of 7 days (spraying the entire plant)
Then, 7 days after the final spraying, the disease degree was investigated.

Further, lettuce obtained in Example 6 which was not treated with the wettable powder was subjected to the same test to calculate the degree of disease, and using this as a control, lettuce was treated in the same manner as in the test example of turfgrass. The control value of the test example was calculated.

Further, for comparison, instead of the 500-fold dilution of the wettable powder of Example 6 above, a 650-fold dilution of the wettable powder obtained in Comparative Example 4 (3.0 × 10 ¹¹ CFU / L), the same test was performed using a commercially available Topzin M wettable powder (main component; thiophanate methyl, manufactured by Nippon Soda Co., Ltd., 667 ppm) to investigate the degree of disease and calculate the control value. The results are shown in Table 10.

[0075]

[Table 10]

(7) Tomato Gray Mold Control Test The control effect against tomato gray mold was tested in a glass greenhouse (variety: Zuiken, test group: 12 strains / group, 3 repeats). The inoculation of tomato Botrytis cinerea was carried out by suspending citrus fruits infected with Botrytis cinerea and cultured in a greenhouse. At the flowering stage of the third fruit cluster of tomato, a 200-fold dilution of the wettable powder obtained in Example 1 (1.0 × 10 ¹² CFU / L) was sprayed three times at 7-day intervals (spray treatment, 200 L / 10a). Diseased fruits and flowers were measured 7 days after the final spraying to determine the disease incidence.

The same test was carried out on the tomatoes which were not treated with the wettable powder obtained in Example 1 above, and the disease incidence was determined. Using this as a control, the tomato test examples were conducted in the same manner as the above-mentioned turfgrass test examples. The control value was calculated.

Further, for comparison, instead of the 200-fold dilution of the wettable powder of Example 1 above, a 200-fold dilution of the wettable powder obtained in Comparative Example 4 (1.0 × 10 ¹² CFU / L) and a commercially available Roniran wettable powder (main component; vinclozolin, manufactured by Sankyo, 667 ppm) were subjected to the same test, and the disease incidence was calculated and the control value was calculated. The results are shown in Table 11.

[0079]

[Table 11]

(8) Test for controlling green mold of kidney bean The control effect on green mold of kidney bean was tested (variety: Satsuki Midori No. 2, test section: 6 m ² / section, no repetition). The attack was spontaneous. At the flowering stage of green beans, a 500-fold dilution of the wettable powder obtained in Example 6 (3.0 x 10
¹¹ CFU / L) was sprayed four times at a rate of 300 L / 10a at 7-day intervals (spray treatment). Seven days after the final spraying, the kidney pods were examined for the presence or absence of disease, and the diseased pod rate was obtained.

The same test was carried out on the green beans obtained in Example 6 which had not been treated with the wettable powder, and the disease pod rate was calculated. The control value of the test example was calculated.

Further, for comparison, instead of the 200-fold dilution of the wettable powder of Example 6 above, a 650-fold dilution of the wettable powder obtained in Comparative Example 4 (3.0 × 10 ¹¹ CFU / L) and a commercially available Roniran wettable powder (main component; vinclozolin, Sankyo Co., Ltd., 667 ppm) were used to perform the same test, and the disease-causing rate was calculated and the control value was calculated. The results are shown in Table 12.

[0083]

[Table 12]

(9) Gray mold disease control test of eggplant (sustainability stability test) The control effect against eggplant gray mold disease was tested in a glass greenhouse (cultivar: 1,000 units, test group: 8 strains / group, 3 repetitions).
The inoculation of the eggplant Botrytis cinerea was carried out by suspending the cultivated eggplant fruits infected with Botrytis cinerea in a greenhouse. At the flowering stage of the eggplant, a 200-fold dilution of the wettable powder obtained in Example 5 (1 × 10 ¹² CFU / L) was sprayed 3 times at 7-day intervals (spraying the entire strain, 300 L / 10a). did. The diseased fruit / flower ratio (%) was calculated 1 week and 2 weeks after the final application.

The same test was carried out on the eggplants obtained in Example 5 which had not been treated with the wettable powder, and the diseased fruit / flower ratio was calculated. This was used as a control in the same manner as in the above test example of turfgrass. The control value of eggplant test examples was calculated.

Further, for comparison, instead of the 200-fold dilution of the wettable powder of Example 5 above, a 200-fold dilution of the wettable powder obtained in Comparative Example 4 (1.0 × 10 ¹² CFU / L), a viable cell suspension obtained in Comparative Example 3 and the culture solution supernatant (antibiotic fraction) obtained in Comparative Example 1 were used to perform the same test, and Calculation and control value were calculated. The results are shown in Table 13.

[0087]

[Table 13]

As is clear from these results, the agricultural / horticultural fungicide composition of the present invention containing spores of a bacterium belonging to the genus Bacillus and a spore germination promoter contains spores of a bacterium belonging to the genus Bacillus. Is more excellent in disease control action than a fungicide composition containing no spore germination accelerator, and the disease control action is equal to or higher than that of conventional synthetic pesticides that easily cause physiological disorders. It also has good fixability and long-term stability as compared with live cells of bacteria belonging to the category II and antibiotics obtained from them.

[0089]

The fungicide composition for agricultural and horticultural use of the present invention is excellent in disease control action by containing spores and spore germination promoters of bacteria belonging to the genus Bacillus, and further has good fixability and long-term stability. . Further, by applying this fungicide composition for agricultural and horticultural use, it is possible to very effectively protect plants from various diseases.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location (C12N 3/00 C12R 1: 125)

Claims (7)

[Claims] 1. A fungicide composition for agricultural and horticultural use containing spores of a bacterium belonging to the genus Bacillus and a spore germination promoter. 2. The fungicide composition for agricultural and horticultural use according to claim 1, wherein the bacterium belonging to the genus Bacillus competes with a plant pathogen. 3. The fungicide composition for agricultural and horticultural use according to claim 2, wherein the bacterium belonging to the genus Bacillus that antagonizes the plant pathogen is Bacillus subtilis. 4. The Bacillus subtilis, the Bacillus subtilis, and the Bacillus subtilis FERM.
P-14647 strain, Bacillus subtilis FERM
The agricultural and horticultural fungicide composition according to claim 3, which is selected from P-14646 strains. 5. The agricultural and horticultural method according to claim 1, wherein the spore content of the bacterium belonging to the genus Bacillus is 0.001 to 99.9 parts by weight based on the total amount of the composition. Disinfectant composition. 6. The agricultural / horticultural germicidal composition according to claim 1, wherein the spore germination promoter is selected from a nutrient substrate, a chelate compound, and an inorganic salt. 7. The agricultural / horticultural fungicide according to claim 1, wherein the content of the spore germination promoter is 0.1 to 99.999 parts by weight with respect to the total amount of the composition. Composition.