FR2467215A1 - NOVEL ANTIBIOTICS, BOTRYTICIDINE A, AND A PROCESS FOR PREPARING THE SAME, AND A FUNGICIDE COMPOSITION FOR AGRICULTURE AND HORTICULTURE CONTAINING BOTRYTICIDINE A AS ACTIVE INGREDIENT - Google Patents

Abstract

The subject of the invention is a novel antibiotic, Botryticidin A, as well as a process for its preparation and a fungicidal composition for agriculture and horticulture containing Botryticidin A as an active ingredient. This new antibiotic, Botryticidin A, is obtained by culturing a microorganism of the Bacillus type. Its infrared absorption spectrum is shown in Figure 2. This antibiotic exhibits an inhibitory effect on the culture of various pathogenic bacteria of plants. (CF DRAWING IN BOPI)

Description

The present invention relates to a novel antibiotic, Botryticidin A,

  as well as a process for its preparation and a fungicidal composition for

  agriculture and horticulture containing Botryti

  cidine A as an active ingredient.

  Botryticidin A of the invention is

  culture of a Botryticidin A producing bacterium belonging to the genus Bacillus and by

  fractionation of the culture broth.

  Among the known antibiotics one can

  cite those produced from the microorgan-

  For example, Bacileucines A and B which are produced from a microorganism belonging to the Bacillus species both serve as active ingredients in a Bacillus genus.

  fungicidal composition for agriculture and horticultural

  and new antibiotics that the plaintiff is the first to develop (see

  U.S. Patent No. 4,181,714).

  It has been discovered that Bacileucines A and B pro-

  have a very potent effect against the major diseases of rice, namely burning and anthracnose of rice and diseases affecting various other

  plants such as gray rot and anthracis

  nose of the cucumber, the black stain of the pear and the stained rot of the vine. It has also been discovered that the new antibiotics Bacileucines A and B have no phytotoxicity to all types of plants and that they do not produce

  no adverse effect on the human body.

  The present invention relates to a novel basic antibiotic, Botryticidin A, produced from a microorganism belonging to the genus Bacillus and having a strong inhibitory effect on the growth of various plant pathogenic bacteria, as well as a method for its preparation and that a fungicidal composition for agriculture and horticulture containing Botryticidin A in

as an active ingredient.

  The novel antibiotic of the invention, Botryticidin A, has a potent effect against

  certain plant diseases, such as the main

  rice diseases, namely rice blight, Helminthosporiosis and rice anthracnose, several diseases affecting vegetables, namely gray mold, anthracnose, white and angular spots

  cucumber, tomato sclerotinia, black

  sweet potato rot, soft rot of Chinese cabbage and several fruit diseases such as black pear blotch disease and angular vine blotches. This antibiotic has no phytotoxicity to other plants and has no adverse effect on the human body

during its application.

  FIG. 1 represents the ultraviolet absorption spectrum of Botryticidin A of the invention and FIG. 2 represents the infrared absorption spectrum.

Botryticidin A. -

  The novel antibiotic of the invention, Botryticidin A and its method of preparation are

described in more detail below.

  The microorganism used in the present invention is capable of producing Botryticidin A and belongs to the genus Bacillus. One example is Bacillussubtilis AJ1316, which has the property mentioned above to produce Botryticidin A. This strain can advantageously

  be used in the process of the invention.

  According to the "Bergey's Manual of Determinative Bacteriology (8th Edition)", Bacillus subtilis AJ1316 (NRRL B-12231) (hereinafter referred to as "sp.AJ1316") described above is a strain belonging to the genus Bacillus subtilis. Sp. AJ1316 has been deposited at the Institute of Fermentation Research, at the Agency for Science and Industrial Technology, Ministry of International Trade and Industry under the heading FERM and with for access number, FERM-P No. 5154 and the Agriculture Research Crop Collection (hereinafter referred to as "NR1L") (1815 North University Street, Peoria, IL, 61604) under NRRL with Accession Number B-12231

  and is deposited with the NRRL with free access to the public.

  The sp. AJ1316 (NRRL B-12231) is an example of a microorganism that can be used in the invention. Not only can natural and artificial mutants of sp. AJ1316 (NRRL B-12231) but also all species belonging to the genus Bacilluset capable of producing Botryticidin A. The method of the invention allows to cultivate a bacterium producing Botryticidin A belonging

  to the Bacillus genus according to a conventional method of

  antibiotics. The culture method is not critical and one can adopt a liquid or solid culture method. In order to advantageously carry out the cultivation on an industrial scale, it is recommended to use a process consisting in inoculating in a broth of culture a suspension of spores or a culture broth of a bacterium producing Botryticidin and to carry out the culture under aeration

and with agitation.

  The nutrient source used in the invention is not particularly critical and any nutrient source commonly used for culturing microorganisms can be used. By way of example, assimilable carbon compounds such as glucose, sucrose, lactose, maltose, starch, dextrin, molasses, glycerol and cellulose can be used as the carbon source. and as nitrogen source, nitrogenous substances such as corn liquor, soybean powder, cottonseed powder, wheat gluten, peptone, meat extracts, yeast extracts can be used. yeast, soy protein hydrolyzate, casein hydrolyzate, ammonium salts and nitrates. If desired, add an anti-foaming agent such as silicone oil, soybean oil, surfactant, etc. The culture temperature of -15 can be chosen in a range which does not prevent the production of Botryticidin A. It is between 20 and 350C and preferably between 25 and 300C. The cultivation time varies depending on the other growing conditions, but is usually between 24 and

  96 hours and preferably between 24 and 48 hours.

  The initial pH of the culture broth is adjusted to about 6.0-6.8. It is desirable to complete the culture when the potency of Botryticin A reaches

its highest level.

  Botryticidin A accumulates in the broth in which the bacterium producing Botryticidin A has been so cultivated. As a result, the cells are removed from the culture broth to separate and collect Botryticidin A. An example of the method for separating and purifying Botryticidin A is described below: The sp. AJ1316 (NRRL B-12231) at 270 ° C. for 24 hours in a culture medium on an inclined plane (YMG culture medium) containing 0.3% of yeast extract, 0.3% of malt extract,

  0.5% of polypeptone, 1.0% glucose and 1.5% agar

  agar. The strain cultured in the inclined medium mentioned above is inoculated into 100 ml of a culture broth (sterilized at 120 ° C. for 20 minutes), initial pH = 6.2) containing 2% starch, 2% glucose, 2 % soybean powder and 0.05% antifoaming agent (main ingredient: polypropylene glycol) placed in a Sakaguchi bottle, then grown at 27 ° C for

24 hours (pre-seeding).

  The seed is then cultured and then the main crop is cultivated to give the culture broth containing Botryticidin A. Inclined culture sp. AJ1316 (NRRL B-12231) YMG culture medium, 27 C, 24 h Pre-inoculation In a Sakaguchi flask, capacity: 100 ml, 27 C, 24h (seed volume: 2%) binds Seeding Quantity: 20 l, 27 C, 24 h. Iagitation: 350 rpm

aeration: 1/2 v / v / min.

  internal pressure: 0.5 kg / cm2, Ir t (seed volume 2%) Main cultivation Quantity: 300 ú, 27 C, 24-48 h stirring: 300 rpm

aeration: 1/2 v / v / min.

  internal pressure: 0.5 kg / cm2 Broth culture containing Botryticidin A

  The broth of culture containing Botryti

  dine A thus obtained is subjected to centrifugation for

  eliminate the cells. The liquid is passed over

  resulting in a column packed with an adsorption resin such as a Dowex-50 W cation exchange resin (H type) (trade name). Distilled water is passed through the column until

  that no organic compounds remain in the eluate.

  The adsorbed substances are eluted with a 1N aqueous solution of NH 4 OH and then with distilled water until the pH value reaches 8. The entire eluate with a pH greater than 8 is collected and concentrated. below 400C to remove ammonia. The resulting concentrate is passed through a column packed with a substance such as a CM-Sephadex C-25 ion exchange resin (trademark) and then distilled water is passed through this column to completely remove the substances. The adsorbed substances are then eluted using a 0.1 M-1.0 M linear gradient of NaCl and each eluted fraction is subjected to in vitro tests according to the paper disc method. active in the final fraction which is eluted with about 0.68 M NaCl.

  active fraction, then it is desalted dialytically.

  The solution thus dialyzed is again lyophilized to give Botryticidin A as a white powder. The various operations described above can be represented by the following flowchart. Culture broth Cells are removed by centrifugation Adsorption on Dowex 50 W (H eluted with NH 4 OH) Eluate (pH: less than 8) Concentrated at 40 C under vacuum to remove NH3 Adsorption Concentrate on CM-Sephadex C-25 Eluted with a linear gradient of Q, 1 to 1.0 M NaCl Eluat Active Fraction (eluted with approximately 0.68 M NaCl) Lyophilization (concentrated up to a volume of 41-of 1/10) Dialysis Freeze-drying 1, White powder (Botryticid A) Botryticidin A thus obtained is a

  new antibiotic with the physical properties

following biological and chemical

  Physicochemical properties of Botryticidin A (1) Elemental composition:

C = 53.34% H = 7.55%

N = 24.32% 0 = 14.77%

  (2) Molecular weight (estimated from amino acid analysis): 6,400 (3) Melting point: Since Botryticid A is a high molecular weight peptide, its melting point can not be reached.

to be determined.

  (4) Specific rotatory potency: {a} 25: + 1.10 (C = 0.02, in 0.8M D NaCl solution)

2467215.

  (5) Ultraviolet Absorption Spectrum: The ultraviolet absorption spectrum of Botryticidin A, determined in a 0.9 M NaCl solution, is shown in FIG. 1. The maximum absorption is observed at 207 nm (E0'005%: 0.3). 1 cm (6) Infrared Absorption Spectrum: The infrared absorption spectrum of Botryticidin A is shown in Figure 2. Some specific absorption bands are observed for wave numbers of 1630, 1210, 1160, 1110, 1090 ,

1040, 980, 730 cm.

(7) Solubility in solvents:

  Soluble in water but hardly

  in conventional organic solvents such as methanol, ethanol, butanol, ethyl acetate, ether, chloroform, benzene and the like. (8) Staining reactions: Positive reaction to biuret and ninhydrin

  but negative to Foline and Molisch's reaction.

  (9) Basis, acidity or neutrality:

basic substance.

(10) Color: -

White, amorphous powder.

  (11) Thermal stability: - Crude Botryticidin A (concentrated before adsorption on CM-Sephadex C-25 or in solution in NaCl (concentration: 200 ppm) above 0.6M, is stable at a temperature ranging from room temperature at 100 C, but the antibiotic activity of the

  raw substance decreases when it is desalinated.

  (12) Amino acid composition: The amino acid composition of Botryticidin A, determined using an automatic amino acid analyzer (Model NIPPON-DENSHI

JLC-6AH) is given below.

  Amino acid Number of molecules Serine 11 Leucine 10 Glycine 9 Glutamic acid 7 (including glutamine) Phenylalanine 5 Alanine 4 Aspartic acid 3 (including Asparagine) Ornithine 3 Lysine 2 Histidine 2 Valine 2 Threonine 2 Arginine 1 Isoleucine 1 Biological properties of Botryticidin A (1) Antimicrobial spectrum: The antimicrobial spectrum of Botryticidin A against various plant pathogenic bacteria determined by the paper disc method (the concentration of Botryticidin (active fraction with approximately 0.68M of NaCl) is 10 ppm) as shown in the following table

  Pathogenic bacteria Size of the circle of

  growth growth (mm) Botrytis cinerea (causing gray cucumber rot) Colletotrichum lagenarium (causing cucumber anthracnose)

2467215;

  Alternaria kikuchiana 25 (causing black spot disease of the pear) Ceratocystis finbriata 40 (causing black spot disease of sweet potato) Glomerella cingulata 42 (causing vine rot of the vine) Rhizoctonia solani 25 (causing canker blight) rice sheath) Pyricularia oryzae 35 (causing burning of rice) In addition, Botryticid A has a slight antimicrobial effect against other bacteria such as staphylococcus aureus, Streptococcus

  epidermis, Micrococcus fulvus and Sarcina lutea.

(2) Toxicity:

  The acute toxicity of Botryticidin A vis-

  mice is such that they are not killed by abdominal or oral administration of mg / kg and therefore, it has been confirmed that the

  Botryticidin A has very low toxicity.

  Since Botryticidin A is a high molecular weight peptide, it can be compared to an enzyme. The substances belonging to the groups of enzymes have an enzymatic activity, that is to say that they have a specific activity acting only with respect to the particular structure of the substrate to be treated, whereas Botryticidin does not has no effect of this type. Substances that belong

  antibiotics are metabo-

  and inhibit the growth of other microorganisms, and their molecular weight or degree of activity is not limited. Although Botryticidin A is a high molecular weight substance, it has antibiotic activity as described above. Consequently, given the physico-chemical and biological properties of Botryticidin A,

  it is reasonable to classify it among antibiotics.

  The physico-chemical and biological properties

  described below are compared with

  those of known antibiotics described in the literature.

  and, in particular, antibiotics

  from microorganisms belonging to the genus Bacillus.

  First, Bacillomycin differs significantly from Botryticidin A in its

  molecular weight (Bacillomycin has a molecular weight

  less than 1000). Mycobacillin differs

  Botryticidin in that the latter

  is a circular polypeptide having a molecular weight

  of 1,800 and is soluble in alcohols (see Nature, 181, 134 (1958) and Biochemical, J. 121, 839 (1971)). In addition, the substance Fraction A (see Mira Sen & P. Nandi, "Isolation of the Active Principles from a Strain of Bacillus subtilis", Indian J. Chem., Vol 1, p 135-136) differs markedly from

  Botryticidin A with respect to its absorption spectrum

  ultraviolet and maximum absorption band (substance Fraction A: 226 nm) and its solubility in various organic solvents). Finally, Bacileucines A and B (see United States Patent No. 4,181,714) also differ from

  Botryticidin A with regard to their molecular mass

  (these two Bacileucines have a molecular weight of 13 000), the ultraviolet absorption spectrum and the maximum absorption band in the ultraviolet (the Bacileucines A and B have maxima respectively at 273 and 260 nm), the stability (Bacileucines A and B lose 50% of their antimicrobial activity when kept at 60 ° C. for about 10 minutes at pH 7.2), the composition of the amino acids, and that Bacileucines A and B are B are both

acidic substances.

  Therefore, it is impossible to find a known substance that can be considered as - identical to the Botryticidin A of the invention. It has been concluded that Botryticidine.A is a new antibiotic. As mentioned above, the antibiotic of the invention, Botryticidin A, differs from all antibiotics known from the point of view of its physicochemical properties and has never been

described in the literature.

  The invention also relates to a composition

  fungicidal formulation for agriculture and horticulture containing the new antibiotic described above

as an active ingredient.

  As agricultural-use and horticidal fungicides

  In the past, products containing a heavy metal compound such as

  that preparations of copper, mercury and

  senic, organic chemicals based

  chlorine and phosphate. However, all these fungi

  Classical agricultural and horticultural crops are toxic to animals and the human organism. In addition, they contaminate the soil and remain in the environment for prolonged periods while exerting a detrimental influence on animals and plants. As a result, environmental pollution by these chemicals poses

2467215.

  currently a serious social problem and the use

  of these products is prohibited or restricted.

  However, various plant diseases such as rice diseases increase in virulence as the number of usable chemicals decreases, so that it became essential in the art to develop a new agricultural product. having a powerful effect against plant diseases and presenting

  a high degree of security. Consequently, the request

  deresse undertook research with the aim of

  develop an agricultural chemical of this type.

  It resulted in the discovery that the new anti-

  biotic mentioned above, Botryticidin A,

  allowed to act very effectively against the main

  diseases of rice, namely rice blight and rice sheath canker, and against diseases of various other plants such as gray mold, white, angular spots and cucumber anthracnose, black spots of the pear, the an-thracnose of the vine, the disease of tomato stains, the black-rot of the sweet potato and the soft rot of the Chinese cabbage, these effects being confirmed by tests of spray. It was further discovered that the new antibiotic showed no phytotoxicity to anyone.

  what kind of plants and what did it produce -

  no adverse effect on the human body. On the basis of these discoveries, the plaintiff

  point the agricultural and horticultural fungicide of the invention.

  When Botryticidin A of the invention is used as an agricultural and horticultural fungicide, according to techniques commonly used to make ordinary agricultural chemicals, it may be formulated as an optical preparation such as granules, dust, an emulsifiable liquid, a wettable powder, tablets, an oil, a spraying liquid or a fumigant using a solid support, a liquid carrier or an appropriate emulsifier dispersant commonly used in the art. Examples of solid carriers are clay, kaolin, bentonite, acidic clay, diatomaceous earth, calcium carbonate, nitrocellulose, starch, gum arabic and mixtures thereof. of these and as liquid carriers, there may be mentioned for example water, methanol, ethanol, acetone, dimethylformamide, ethylene glycol and mixtures thereof. In addition, it may be convenient to incorporate additives commonly used in the manufacture of agricultural chemicals, such as sulfuric acid esters of alcohols.

  polyalkyl aryl alkyl ethers.

  oxyethylene, polyalkyl aryl alkyl ethers,

  ethylene glycol, alkyl aryl sorbitan monolaurates,

  alkyl aryl sulphonates, alkyl sulpho

  alkyl aryl sulphonic acid salts, quaternary ammonium salts, polyalkylene oxides and

  mixtures thereof. A report should be used

  mixing port of the active ingredient from about 10 to about 90% in the case of an emulsifiable liquid or a wettable powder and from about 0.1 to about 10% in the case of a powder or powder. an oil. It is clear that the mixing ratio of the active ingredient can

  be modified according to the purpose of the intended application.

  The fungicide of the invention may further contain other fungicides, herbicides, insecticides, fertilizers or soil modifying agents as needed. The following nonlimiting examples are given by way of illustration of the invention. Unless otherwise indicated, the terms "%" and "parts" respectively mean weight percentages and parts by weight in the remainder of this specification and in the

claims.

* Example 1

  (Botryticidin A) Inoculate sp. AJ1316 (NRRL B-12231) described above in an inclined culture medium containing 0.3% yeast extract, 0.3% malt extract, 0.5% polypeptone, 1% glucose and 1, 5% agar-agar

  and cultivated at 27 C for 24 hours.

  The strain cultured on an inclined medium is then inoculated in 100 ml of a culture medium placed in a Sakaguchi flask (sterilization: 120 C, 20 min, initial pH: 6.2) containing 2% of starch, 2% of

  glucose, 2% of soybean powder and 0.05% of anti-

  foam (main ingredient: polypropylene glycol) and subjected to a pre-seeding

  at 27 ° C for 24 hours (seed volume: 2%).

  The culture of the seed is carried out at 27 ° C. for 24 hours under the following conditions:

  amount; Stirring: 350 rpm, aerator

  tion: 1/2 v / v / min and internal pressure 0.5 kg / cm 2.

  The main culture is then carried out at 27 ° C. for 48 hours under the following conditions: quantity: 300 l, stirring: 300 rpm, aeration: 1/2 v / v / min and internal pressure 0.5 kg / cm 2, this which gives 300 liters of a culture medium containing Botryticidin A. One liter of the culture medium thus obtained is subjected to centrifugation at 10,000 rpm for 30 minutes to eliminate the cells. column packed with 500 g of Dowex-50W (type H +) and this volume of distilled water is passed three times through the column to wash off non-adsorbed organic substances. The adsorbed substances are eluted with one liter of an aqueous NH 4 OH solution and then eluted with distilled water until the pH of the eluate becomes less than 8. The whole of eluate whose pH is above 8 and concentrated at 40 ° C under vacuum to eliminate

  ammonia. One liter of the concentrate is obtained.

  This concentrate is passed through a column packed with CM-Sephadex C-25 (Trade Mark) and then distilled water is passed through the column to completely remove non-adsorbed substances. The adsorbed substances are eluted with a linear gradient

  from 0.1 to 1 M NaCl at a flow rate of 3 ml / min. In deter-

  the anti-microbial spectra of each of the

  fractions according to the paper disc

  10 ppm, pathogenic organism: Botrytis cinerea), it was found that the active substance was contained in the final fraction eluted by

about 0.68M NaCl.

  The active fraction is lyophilized to a concentration of 1/10 by volume and dialyzed through a Visking tube to desalt it. The solution is freeze-dried

  dialyzed and desalted, resulting in 10 mg of Botrytic-

  dine A in the form of a white powder.

Example 2

  (wettable powder) A mixture of 10 parts of Botryticidin A (concentrated before adsorption on CM-Sephadex C-25), 5 parts of lauryl sulfate, is prepared.

  sodium, 2 parts of a sodium condensate dinaphthyl-

  methane-disulfonate-formaldehyde and 83 parts of clay and is sprayed to give 100 parts

wettable powder.

Example 3

  (emulsifiable liquid) A mixture of 8 parts of Botryticidin A (concentrated before adsorption on CM-Sephadex C-25), 10 parts of ethylene glycol, 20 parts of dimethylformamide, 10 parts of alkyl dimethylbenzyl ammonium chloride is prepared. and 52 parts of methanol and dissolved, giving 100 parts

an emulsifiable liquid.

Example 4

  (Powder) A mixture of 0.2 part of Botryticidin A (concentrated before adsorption on CM-Sephadex C-25), 0.5 part of calcium stearate, parts of talc and 9.3 parts of clay is prepared. we

  sprays, giving 100 parts of a powder.

Example 5

  (Granules) A mixture of 10 parts of Botryticidin A (concentrated before adsorption on CM-Sephadex C-25), 15 parts of starch, 72 parts of bentonite and 3 parts of sodium lauryl sulfate is prepared.

  and pulverized to give 100 parts of granular

the.

  The effects of agricultural and horticultural fungicide

  of the invention against various plant diseases are described in detail hereinafter with reference to the following tests: Test example No. 1 Test of the effect against gray mold of cucumber

  A wettable powder prepared is diluted

  according to the method described in Example 2 with water to a predetermined concentration and

  sprays the dilution on coconut seedlings. (go-

  Sagami Hanjiro) obtained 15 days after sowing.

  and dry the pulverized chemical

in the air.

  A pathogenic parasite is cultivated causing

2467215;

  Gray mold of cucumber (Botrytis cinerea)

  in a potato plate culture medium-

  glucose-agar-agar and is irradiated with

  black light "Black Light Blue" to induce spore formation. The spores are then suspended in an aqueous solution containing 10%

  glucose and 1% yeast extract.

  After drying the sprayed chemical, the young plants are placed in an inoculation box. The suspension is sprayed on the seedlings by means of a spray gun

at a rate of 10 ml per 10 plants.

  The plants thus inoculated are maintained at a temperature of 20 ° C. and at a relative humidity of 100% for 4 days, then the diseases are observed.

appearing in plants.

  The degree of effectiveness was calculated according to the following procedure: Disease index Extent of disease observed o Uncommonly observed leaf effi ciency of less than 10%

at 20%

at 30%

at 40%

  greater than 40% (sum of the disease index cacity (%) in the treated zone X 100 -sai = e of the disease index k in the untreated zone The results obtained are given in the table

table 1.

TABLE 1

  Note (1) Methyl-1- (butylcarbamoyl) -2-benzimidazole carbamate Test example No. 2 Test of efficacy against anthracnose of cucumber An emulsifiable liquid prepared by the process described in US Pat. Example 3 until a predetermined concentration is obtained and the dilution is sprayed on cucumber seedlings (Sagami Hanjiro variety) obtained 15 days after seeding and the chemical is dried

sprayed in the air.

  A pathogenic parasite causing anthracnose of the cucumber (Colletotrichum lagenarium) is cultivated

  in a potato plate culture medium-

  agar-agar and the spores thus cultured are suspended in water. The spore concentration in the suspension is adjusted to observe approximately spores in the field of a working microscope

with a magnification of 150.

  After drying the sprayed product,

  the seedlings are introduced into a tin box

  lation. The suspension is sprayed on youth Concentration in

  Dilution of Pulse- Degree of Effi- Phytoto-

  Testarized substance (ppm) cacity xicity Botryticidin A 100 100 not observed Botryticidin A 100 100 not observed Botryticidin A 50 10099 not observed Botryticidin A 1025 99 not observed Botryticidin A 10 90 not observed Botryticidin A 10 90 'not observed Benlate (1) 250 95 unobserved Control 0 unobserved plants using a spray gun at a rate of 10 ml per 10 plants. We keep the seedlings

  thus inoculated into the inoculation box at a time

  20 C and a relative humidity of 100% for 24 hours, then introduced into a greenhouse using natural light and kept there for 4 days. We then observe the

  diseases developed in these plants.

  The degree of effectiveness is calculated from the following formula sum of disease spots Efficacy (%) = (_ in the treated area 100s of disease spots in the untreated area - The results obtained are given in the

table 2.

TABLE 2

  Note (2) Tetrachloroisophthalonitrile Test Example 3 - Test of Rice Burning Efficiency Ten rice plants (Jukkoku variety) are grown in a pot and in the four-leaf stage, a powder is diluted with water Wet-able prepared by the process

  described in Example 2 until a concentration of

  Pre-treatment and the dilution is sprayed on Concentration in Substance Tested for Pulse Dilution Efficiency Level Reagent (ppm) Cactivity (%) Botryticidin A 100 99 Not Observed Botryticidin A 50 95 Not Observed Botryticidin A 25 88 Not Observed Botryticidin A Unobserved Daconil (2) 1250 95 The plants were not observed with a spray gun at a rate of 50 ml per pot. Once the chemical is sprayed and dried, a suspension prepared by suspending spores of a pathogenic bacterium causing burning of the rice (Pyricularia oryzae) grown in a culture medium containing a suspension of water is uniformly sprayed into water. rice bark (containing 3 g of rice husks, 0.01 g of yeast extract powder, 0.2 g of sucrose, 0.05 g of starch and 5 ml of water) and inoculates on the rice plants in an inoculation box. After having kept the pots in the inoculation box maintained at a temperature of 25 ° C. and at a relative humidity of 100%, the plants are removed from the inoculation box and maintained

  at room temperature. When we observe the

  after 5 to 7 days after inoculation.

  the number of disease spots per pot. The degree of effectiveness is calculated using the method described in Test Example No. 2.

  The results obtained are given in Table 3.

TABLE 3

  Test product Concentration of product Efficacy degree Phytotoxicity Product tested in the liquid Defect of phytotoxicity sprayed (ppm) Botryticidin A 100 100 not observed Botryticidin A 50 100 not observed Botryticidin A 25 97 not observed Botryticidin A 10 95 not observed Blastitis S ( 3) 10 96 not observed Note (3) Blasticidin Benzylaminobenzenesulfonate S. Test Example No. 4 Tomato stem rot efficacy test A wettable powder prepared according to Example No. 2 was diluted to a pre-determined concentration and 50 ml of the dilution is sprayed on young tomato plants (Fukuju variety) at the seven- or eight-leaf stage after seeding. After

  have allowed the pulverulent chemical to air dry

  checked, a sample is applied and inoculated

  colony obtained by cutting out a peripheral zone

  colony of Sclerstinia sclerotiorum by means of a cork drill (about 5 mm diameter)

  a predetermined part of a leaf of the plant.

  The inoculated colony was grown in a medium

* potato-glucose-based plate culture

  classic agar. Inoculated tomato plants are maintained at a temperature of 250C and a relative humidity above 95% for 3 days, then the size of the disease spots is measured. The degree of effectiveness is calculated in accordance with

next formula -

  Somne stain sizes of Efficacy (%) - disease in treated area d 100

  knocks on the sizes of

  in the untreated zone The results obtained are given in the

table 4.

2467215;

TABLE 4

  Note (4) {1,2-Phenylenebis (iminocarbonothioyl)} biscarmic acid diethyl ester Test example No. 5 Test of effectiveness against soft rot of Chinese cabbage A wettable powder prepared by the process is diluted of Example 2 to a predetermined concentration and 50 ml of this dilution is sprayed on seedlings of Chinese cabbage (Nozaki variety

  N 2) grown for 13 days after seeding.

  Three seedlings are used per test area. After air-drying the sprayed material, a seed suspension prepared by suspending cells from a pathogenic bacterium causing soft rot is inoculated and sprayed on the seedling of Chinese cabbage using a spray gun. certain vegetables (Erwinia carotovora) grown in a culture medium

  Suwa at 280C for 20 hours while shaking.

  After inoculation, the seedlings are kept in an inoculation box at a temperature of 28 ° C. and a relative humidity of greater than 95%.

  for 3 days, then we observe the extent of the disease.

J Concentration of the pro-

  Tried product in the liquid Level of effi- Phytotoxicity sprayed (ppm) cacity (%) Botryticidin A 100 99 not observed Botryticidin A 50 90 not observed Botryticidin A 25 87 not observed Topsin M (4) 1000 85 not observed

Look - -0 -

2467215:

  The degree as follows: Efficacy Sickness Index is calculated from the Extent of Disease on Efficacy (%) = leaves not observed very slightly detectable slightly detectable about 1/4 of the surface about 1/3 of the surface about 1/2 of the surface fully attacked somer indexes of disease in the treated area sum of disease indices in the untreated area The results obtained are given in the

Table 5.

TABLE 5

Concentration of the pro-

  Tried product in liquid Level of effi- Phytotoxicity sputtered (ppm) cacity (%) Botryticidin A 100 81 not observed Botryticidin A 50 75 not observed Botryticidin A 25 69 not observed Agret (5) 100 68 not observed Tamoin 0 not observed Note (5) Streptmycin Example of test No. 6 Test on the effect on white of cucumber A wettable powder prepared X 100 is diluted according to Example 2 to a predetermined concentration and 50 ml of this dilution is sprayed on young cucumber seedlings (Sagami variety

  Hanjiro) grown for 14 days after seeding.

  Three young plants are used per test area. The sprayed product is allowed to dry. A pathogenic bacterium causing white cucumber (Sphaerotheca fuliginea) is inoculated on a developed cucumber leaf. At the end of the day after the inoculation,

  a colony of this bacterium has formed. We sprayed

  rise and inoculate on young cucumber plants a suspension of spores taken from this colony

  (the concentration of spores being 1 x 105 spores / ml).

  The plants thus inoculated are introduced into a greenhouse made of vinyl film at a temperature of

  25 to 30 ° C and held there for 11 days.

  Then we count the number of disease spots on

leaves.

  The degree of effectiveness is calculated according to

in accordance with Test Example 2.

  The results obtained are given in the

table 6.

TABLE 6

Rating (6) Quinomethionate

Concentration of the pro-

  Tried product in liquid Level of effi- Phytotoxicity sprays (ppm) cacity (%) Botryticidin A 100 90 not observed Botryticidin A 50 82 not observed Botryticidin A 25 79 not observed Morestan (6) 60 85 not observed

Witness- 0-

  Test Example No. 7 Efficiency Test against Cucumber Angular Blots A wettable powder prepared according to Example 2 was diluted to a predetermined concentration and 50 ml of this dilution was sprayed on both sides of the leaves. cucumber (Suyo variety) at the two-leaf stage. After allowing the spray to air dry, the leaves of cucumber seedlings are sprayed and inoculated with a suspension of spores of a pathogenic bacterium causing the angular spots of the cucumber.

  cucumber (Pseudomonas lachrymans) obtained by

  culture for 24 hours. The young plants thus inoculated are kept in a greenhouse at a temperature of 25 ° C for 2 days, then introduced into

  another greenhouse using natural light.

  The extent of the disease is then observed.

  The degree of effectiveness is calculated according to Test Example No. 5. The results obtained are

given in Table 7.

TABLE 7

  Concentration of the product Test product eluted in the sprayed liquid (ppm) cacity%) Botryticidin A 200 99.not

Botryticidin A 100 85 r -o abser-

  Botryticidin A 50 80 norn observed Agret 50 70 non obsev t Witness O 0

__________________.

  Comments Based on the results of the tests described above, it appears that the fungicide containing Botryticidin A of the invention as an ingredient

2467215.

  active, has a very high degree of efficiency vis-à-

  different plant diseases and has no phytotoxicity, ie it can be used on a large scale safely to control many diseases.

Claims (10)

  1. New antibiotic designated under the name Botryticidin A, characterized in that it has the following physicochemical and biological properties: 1) Elemental composition: C = 53.34% H = 7.55% N = 24.32% 0 = 14.77%   2) Molecular weight: 6,400 3) Specific rotatory power: [α 25: +1.10 (C = 0.02, in 0.8M NaCl solution D) 4) Ultraviolet absorption spectrum: The spectrum of Ultraviolet absorption of Botryticidin A, determined in 0.9 M NaCl solution, is shown in Figure 1. The maximum absorption is observed at 207 nm (E 0.005%: 0.3); 1 cm) Infrared Absorption Spectrum: The infrared absorption spectrum of Botryticidin A is shown in Figure 2. Particular absorption bands are observed at wave numbers 1630, 1210, 1160, 1110, 1090, 1040 980, 730 cm -1; 6) Solubility in solvents: Soluble in water but hardly soluble in conventional organic solvents such as methanol, ethanol, butanol, ethyl acetate, ether, chloroform, benzene, etc ...; 7) Staining reactions: Positive reaction to biuret and ninhydrin but negative reaction to Foline and Molish reaction; 8) Basicity, acidity or neutrality: Basic substance; 9) Color White amorphous powder; ) Thermal stability: Botryticidin A crude (concentrated, before adsorption on CM-Sephadex C-25) or Botryticidin A (concentration: 200 ppm) in NaCl solution above 0.6 M, is stable at temperature ranging from room temperature to 100 C, but its antibiotic activity decreases when desalting Botryticidin A crude; 11) Amino acid composition: The amino acid composition of Botryticidin A is shown below: Amino acid Number of molecules Serine 11 Leucine 10 Glycine 9 Glutamic acid (including glutamine) 7 Phenylalanine 5 Alanine 4 Aspartic acid (y includes asparagine) 3 Ornithine 3 Lysine 2 Histidine 2 Valine 2 Threonine 2 Arginine 1 Isoleucine 1 12) Antimicrobial spectrum: Botryticid A has specific antimicrobial activity against various plant pathogenic bacteria, such as Pyricularia oryzae, Rhizoctonia solani, Botrytis cinerea, Colletotrichum lagenarium, Alternaria kikuchiana, Glomerella cingulata, Ceratocytis finbriata, etc ...   2. Process for the preparation of a novel antibiotic, namely Botryticidin A, characterized in that a Botryticidin A producing bacterium belonging to the genus Bacillus is cultivated and in that the new antibiotic is separated and extracted from broth culture.   3. Process according to claim 2, characterized   in that the bacterium producing Botryticidin A belonging to the genus Bacillus is Bacillus subtilis AJ1316 (NRRL B-12231).   4. Process according to claim 3, characterized   in that the Botryticidin A producing bacterium belonging to the genus Bacillus is cultured in an inclined culture medium, in a pre-seeding culture broth, in a seed culture broth and finally in a main culture broth. .   5. Process according to claim 4, characterized   in that the separation of Botryticidin A is carried out by the following operations: a) centrifugation of the culture broth containing Botryticidin A to thereby eliminate the cells, b) Separation of Botryticidin A from the supernatant liquid by column chromatography, and c) purification of Botryticidin A by lyophilization and dialysis.   6. Agricultural and horticultural fungicidal composition containing Botryticidin A as a as an active ingredient.   7. A fungicidal composition for agricultural and horticultural use according to claim 6, characterized in that   that it is in the form of a wettable powder containing   10 to 90% by weight of the active ingredient.   8. fungicidal composition for agricultural and horticultural use according to claim 6, characterized in that it is in the form of an emulsion containing 10 to % by weight of the active component.   9. A fungicidal composition for agricultural and horticultural use according to claim 6, characterized in that it is in the form of a powder containing 0.1 to 10% by weight of the active component.   10. fungicidal composition for agricultural and horticultural use according to claim 6, characterized in that it is in the form of ganulés containing 0.1 to 10% by weight of the active component.