GB2061284A - The antibiotic, Botryticidin A, a method for production thereof, an agricultural or horticultural fungicidal composition comprising Botryticidin A as active ingredient and anti-fungal crop protection method - Google Patents

Abstract

A basic antibiotic polypeptide, Botryticidin A is produced from a microorganism belonging to the genus Bacillus and has a characteristic inhibitory effect on the growth of various pathogenic fungi which attack plants. A process for producing the antibiotic, agricultural and horticultural fungicidal compositions comprising the antibiotic and the use of such compositions in anti-fungal plant protection are described.

Description

SPECIFICATION The antibiotic, Botryticidin A, a method for production thereof, an agricultural or horticultural fungicidal composition comprising Botryticidin A as active ingredient and anti-fungal crop protection method This invention relates to a novel antibiotic, Botryticidin A, to a method for the production thereof, to agricultural and horicultural fungicidal compositions comprising Botryticidin A as an active ingredient thereof and to an anti-fungal crop protection method.

A number of antibiotics have hitherto been produced by culturing microorganisms belonging to the genus Bacillus. For example, the antibiotics Bacileucines A and B are produced from a microorganism belonging to Bacillus sp., and are both active as agricultural and horticultural fungicides (see United States Patent No.4181714). It has been found that Bacileucines A and B, are excellent fungicides in respect of fungal diseases affecting rice, particularly blast and sheath blight, as well as and in respect of other fungal diseases of various other plants, such as grey mould and anthracnose in cucumber, black spot on pears and ripe rot on grapes. It has further been found that the antibiotics, Bacileucines A and B have no phytotoxicity with respect to any kind of plant treated therewith and have no adverse effect on the human body.

According to one aspect of the present invention, there is provided a basic antibiotic, Botryticidin A having characteristic physical, chemical and biological properties to be set out herein.

This invention also provides a method for the production of Botryticidin A, which comprises culturing a Botryticidin A-producing bacterium belonging to the genus Bacillus and separating and collecting the Botryticidin A from the culture medium.

A microorganism belonging to the genus Bacillus has a characteristic inhibition activity on the growth of various pathogenic bacteria of plants and is a useful fungicide for use in agriculture and horticulture.

More particularly, the novel antibiotic of the present invention, Botryticidin A, has an excellent action in control of plant diseases such as the main diseases of rice, namely blast, Helminthosporium leaf spot and sheath blight, several vegetable diseases, namely grey mould, anthracnose, powdery mildew and angular spot in cucumber, tomato stem rot, sweet potato black rot, Chinese lettuce soft rot as well as several diseases of fruit, namely pear black spot and angular spot on grapes. The antibiotic shows phytotoxicity and has no adverse effect on the human body when ingested.

Hence this invention also provides a fungicidal composition comprising Botryticidin A in association with a non-phytotoxic carrier therefor and a method of protecting an agricultural or horticultural crop against fungal attack which comprises applying to the crop such a fungicidal composition.

Any one of a number of microorganisms belonging to the genus Bacillus may be employed to produce Botryticidin A. Of particular value is Bacillus subtllis AJ 1 31 6 and the method of the present invention for producing Botryticidin A will be described primarily with respect to the use thereof.

The above-mentioned Bacillus subtills At 1316 (NRRL B-1 2231) (hereinafter referred to "sp. AJ 1316") is a strain belonging to Bacillus subtilis as described in "Bergey's Manual of Determinative Bacteriology (8th edition)". The strain sp. AJ 1316 has been deposited with The Fermentation Research Institute, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (FERM) under access number FERM-P No. 51 54 and with The Agricultural Research Culture Collection (hereinafter referred to as "NRRL") (1815 North University Street, Peoria, Illinois 61604) under NRRL access number B-1 2231. The strain is on deposit with NRRL in an unrestricted deposit permitting the public full access to the culture.

The strain sp. AJ 131 6 is but one example of microorganisms that can be used in the method of the present invention. Not only can natural and artificial mutants of sp. AJ 1316 be used but also all species belonging to the genus Bacillus and having the ability to produce Botryticidin A.

In carrying out the method of the present invention, Botryticidin A-producing bacteria belonging to the genus Bacillus may be cultured by any conventional method for producing antibiotics. The culturing mode is not particularly critical and either liquid culturing or solid culturing may be adopted. In order to perform culturing industrially particularly effectively it is recommended that a method be used in which a culture medium is inoculated with a spore suspension or culture medium of a Botryticidin A-producing bacterium and that the culturing is carried out under aeration and agitation.

The nutritional source that is used in the method of the present invention is not particularly critical, and any of the nutritive sources customarily used for culturing microorganisms may be used. For example, any carbon compounds that can be assimilated, can be used as the carbon source, for example glucose, sucrose, lactose, maltose, starch, dextrin, molasses, glycerin and cellulose, and any nitrogencontaining substances can be used as the nitrogen source, for example corn steep liquor, soybean powder, cotton seed powder, wheat gluten, peptone, meat extract, yeast extract, yeast, soybean protein hydrolysate, casein hydrolysate, ammonium salts and nitrates. If desired, a defoaming agent such as a silicone oil or soybean oil, and a surface active agent may be added to the culture medium.

The culturing temperature may be appropriately set to be within a range of temperatures at which Botryticidin A can be produced. The temperature used will generally range from 200 to 350C, preferably from 250 to 300C. The culturing time will depend on the other culturing conditions and will normally be from 24 to 96 hours, preferably from 24 to 48 hours. The initial pH of the culture medium is preferably from 6.0 to 6.8. The culturing is suitably ended at the time when the potency of Botryticidin A reaches its highest level.

Botryticidin A is accumulated in the medium in which the Botryticidin A-producing bacterium has been thus cultured. Accordingly, cells are removed from the culture medium prior to separating and collecting Botryticidin A from the culture medium.

One example of a procedure for producing, separating and purifying Botryticidin A is set out in Example 1 hereinafter.

This example shows that when producing Botryticidin A using the preferred strain sp. AJ 1 31 6 (NRRL B-I 2231) it is preferred that the bacterium be cultured, in turn, in a slant culture medium, a preseed culture medium, a seed culture medium and finally a main culture medium.

Moreover the Botryticidin A is preferably recovered from the main culture medium by the following steps: (a) centrifuging the culture medium containing Botryticidin A thereby to remove cells, (b) separating Botryticidin Afrom the supernatant by column chromatography, and (c) purifying the separated Botryticidin A by lyophilisation and dialysis.

Botryticidin A obtained by the method of the invention has the following physical, chemical and biological properties.

Physical/Chemical Properties of Botryticidin A (1) Elemental Analysis: C = 53.34%, H = 7.55%, N = 24.32%, 0 = 14.77%.

(2) Molecular Weight (based on its amino acid assay): 6,400.

(3) Melting Point: Because Botryticidin A is a high-molecular-weight peptide, its melting point cannot be determined.

(4) Specific Rotatory Activity: [Cl:]2D5 +1.1 O (C = 0.02, on 0.8M-NaCl solution).

(5) Ultraviolet Absorption Spectrum: The ultraviolet absorption spectrum of Botryticidin A determined in 0.9 m NaCI solution is shown in Figure 1 of the accompanying drawings. The maximum absorption is observed at 207 nm (E1 5%: 0.3).

(6) Infrared Absorption Spectrum: The infrared absorption spectrum of Botryticidin A is shown in Figure 2 of the accompanying drawings. Specific absorption bands are observed, inter alia at wave lengths of 1630, 1210, 1 160, 1 1 10, 1090, 1040,980,730cm1.

(7) Solubility: Soluble in water but hard to dissolve in common organic solvents such as methanol, ethanol, butanol, ethyl acetate, ether, chloroform and benzene.

(8) Colour Reactions: Positive to biuret reaction and ninhydrin reaction but negative to Folin reaction and Molisch reaction.

(9) Basic, acid or neutral character: Basic substance.

(10) Physical appearance: White, amorphous powder.

(11) Thermostability: Crude Botryticidin A (concentrate before adsorption on CM-Sephadex C-25 in procedure of Example 1 - Sephadex is a Registered Trade Mark) or as purified substance in a concentration of 200 ppm in NaCI solution above 0.6M shows it to be stable at a temperature ranging from ambient temperature to 100 C, but having its antibiotic activity reduced when the crude Botryticidin A is subjected to deionisation.

(12) Amino Acid Composition: Amino acid composition of Botryticidin A determined by an automatic amino acid analyser (Model NIPPON-DENSHI JLC-6AH) is as follows: Amino Acid Number of Molecules Serine 11 Leucine 10 Glycine 9 Glutamic acid 7 (inclusive of Glutamine) Phenyl alanine 5 Alanine 4 Aspartic acid 3 (inclusive of 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 with respect to various pathogenic fungi affecting plants, as determined by paper disc method [Botryticidin (active fraction eluted with about 0.68M NaCI) at concentration of 10 ppm] is as shown below.

Size of Growth-inhibiting Pathogenic Fungi Circle (mm) Botrytis cinerea 45 (cause of grey mould in cucumbers) Colletotricum lagenarium 30 (cause of anthracnose in cucumbers) Alternaria kikuchiana 25 (cause of black spot in pears) Ceratocystis finbria ta 40 (cause of black spot in sweet potatoes) Glomerella cingulata 42 (cause of ripe rot in grapes) Rhizoctonia solani 25 (cause of sheath blight in rice) Pyricularia oryzae 35 (cause of blast in rice) Further, Botryticidin A has a slight antimicrobial activity with respect to other fungi including Staphylococcus aureus, Streptococcus epidermis, Micrococcus fulvus and Sarcina lutea.

(2) Toxicity: The acute toxicity of Botryticidin A to mice is such that mice are not killed by abdominal or oral administration of 1,000 mg/kg and therefore, Botryticidin A has been confirmed as to possess very low toxicity.

Since Botryticidin A is a high-molecular-weight peptide, it may be compared with enzymes.

However, an enzyme has a specific activity, with respect to only a specific substrate structure. In contrast Botryticidin has no such narrowly based activity meeting the definition of an antibiotic in being a metabolic product of a microorganism and being active in inhibiting the growth of other microorganisms. Although Botryticidin A is a high-molecular weight substance often unusual for an antibiotic substance, because it has an antibiotic activity as mentioned above because of its biological properties, Botryticidin may reasqnably be classified as an antibiotic.

The above-mentioned physical, chemical and biological properties of Botryticidin A will now be compared with those of known antibiotics disclosed in literature references, especially antibiotics produced from microorganisms belonging to the genus Bacillus. Firstly, Bacillomycin is significantiy different from Botryticidin A with respect to the molecular weight (Bacillomycin has a molecular weight of less than 1,000). Mycobacillin is different from Botryticidin A in that the former is a cyclic polypeptide having a molecular weight of 1,800 and is soluble in alcohols (see Nature, 181, 134 (1958) and Biochemical J. 121,839 (1971). Furthermore the so-called Fraction A substance (see Mira Sen s P. Nandi: "Isolation of the Active Principles from a Strain of Bacillus subtillis", Indian J.Chem., Vol. 1, pp. 1 35-136) is clearly different from Botryticidin A with respect to its ultraviolet absorption pattern and the maximum absorption band (Fraction A substance: 226 nm) and in that Fraction A substance is soluble in various organic solvents. Finally, Bacileucines A and B (see United States Patent No.4181714) are also different from Botryticidin A with respect to molecular weight (both Bacileucines have a molecular weight of 13,000), ultraviolet absorption pattern and maximum absorption band (Bacileucines A and B have maxima at 273 and 260 nm, respectively), thermostability (Bacileucines A and B lose 50% of their antimicrobial activity when maintained at 600C for more than 1 0 minutes at pH 7.2), amino acid constitution and in that both Bacileucines A and B are acidic substances.

Thus, it is impossible to find out any known substance in the literature that can be regarded as being identical with Botryticidin A of the present invention. Accordingly, Botryticidin A was concluded to be a novel antibiotic substance and was named "Botryticidin A".

Hitherto agricultural and horticultural fungicides have contained a heavy metal compound, for example, copper preparations, mercury preparations and arsenic preparations, or contain organic chlorine chemicals or organic phosphate chemicals as active ingredient. However, each of these conventional agricultural and horticultural fungicides is toxic to animals and to the human body.

Furthermore, they contaminate the soil and remain in the environment for a long time to the detriment of both animal and plant life. Accordingly, environmental pollution caused by these chemicals is now a serious social problem and use of these chemicals is generally prohibited or restricted.

However, various plant diseases, for example, diseases affecting rice, are becoming more prevalent as the number of usable chemicals is reduced and, therefore, it has been necessary to develop new agricultural chemicals having both a major effect on plant diseases and which are safe to use.

Experiments which have been conducted have shown the above-mentioned novel antibiotic, Botryticidin A to meet both these criteria. Spraying tests have shown it to be very effective in combating the main diseases affecting rice, namely blast and sheath blight, as well as diseases affecting various other plants, such as grey mould, powdery mildew, angular spot and anthracnose of cucumber, black spot of pears, anthracnose of grapes, stem spot of tomatoes, black rot of sweet potatoes and soft rot of Chinese lettuce. Furthermore, as the above-indicated biological properties show, the novel antibiotic possesses the required lack of phytotoxicity with all kinds of plants and the required lack of adverse effects on the human body.

Insofar as this invention provides a fungicidal composition comprising the above-mentioned novel antibiotic, Botryticidin A as an effective ingredient, such compositions may be of a basic type commonly used in agriculture or horticulture. Thus the composition may take the form of granules, dust, emulsifiable liquid, wettable powder, tablet, oil, spray or fumigant by using an appropriate solid carrier, liquid carrier or emulsifying dispersant customarily used in the art. The solid carrier can be, for example, clay, kaolin, bentonite, acid clay, diatomaceous earth, calcium carbonate, nitrocellulose, starch, gum arabic or a mixture of two or more thereof. The liquid carrier can be, for example, water, methanol, ethanol, acetone, dimethylformamide,ethylene glycol or a mixture of two or more thereof.Furthermore, adjuvants customarily used in the manufacture of agricultural chemicals, such as sulphuric acid esters of higher alcohols, polyoxyethylene alkyl aryl ethers, alkyl aryl polyethylene glycol ethers, alkyl aryl sorbitan monolaurates, alkyl aryl sulphonates, alkyl sulphonic acid salts, alkyl aryl sulphonic acid salts, quaternary ammonium salts, polyalkyleneoxides and mixtures thereof may be appropriately incorporated in the fungicidal compositions. An appropriate content for the active ingredient in the fungicidal compositions is 10% to 90% in the case of an emulsifiable liquid or wettable powder and from 0.1% to 10% in case of a dust or oil. Of course, the content of the active ingredient may be changed according to the intended subject of application.

The fungicidal compositions of the present invention may also additionally comprise other fungicides or herbicides, insecticides, fertilizers or soil modifiers according to need.

The following non-limiting Examples illustrate the present invention. Except where indicated otherwise, "%" and "parts" means "% by weight" and "parts by weight" respectively in the present specification, as well as in the claims which follow.

EXAMPLE 1 (Botryticidin A) The above-mentioned sp. At 1316 (NRRL B-12231) was inoculated into a slant culture medium comprising 0.3% of yeast extract, 0.3% of malt extract, 0.5% of polypeptone, 1% of glucose and 1.5% of agar and cultured at 270C for 24 hours.

The strain grown in the slant was then inoculated into 100 ml of a culture medium contained in a Sakaguchi flask (Sterilization: 1 200C, 20 min., initial pH 6.2) comprising 2% of starch, 2% of glucose, 2% of soybean powder and 0.05% of defoaming agent (main ingredient: polypropylene glycol) and was subjected to pre-seed culture at 270C for 24 hours (seed-volume: 2%).

Seed culture was carried out at 270C for 24 hours under the following conditions: charge: 20 1.

agitation: 350 r.p.m., aeration: 2 v/v min. and internal pressure: 0.5 kg/cm2.

Main culture was then carried out at 270C for 48 hours under the following conditions: charge: 300 1, agitation: 300 r.p.m., aeration: 2 v/v min. and internal pressure: 0.5 kg/cm2. 300 I of culture medium containing Botryticidin A were obtained. One litre of the culture medium thus obtained was centrifuged at 10,000 r.p.m. for 30 min. to remove cells. The supernatant was passed through a column charged with 500 g of cation-exchange resin (Dowex-50 W (H+ type) (Dowex is a Registered Trade Mark, and is the preferred resin) and then three times the supernatant volume of distilled water was passed through the column to wash out non-adsorbed organic substances. Adsorbed substances were eluted with one litre of 1 N NH40H aqueous solution followed by distilled water until the pH value of the eluate was below 9.All the eluate having a pH value of above 8 was collected and concentrated at 400C under vacuum to remove ammonia. One litre of concentrate was obtained.

The concentrate was passed through a column charged with CM-Sephadex C-25 (Sephadex is a Registered Trade Mark) and then distilled water was passed through the column to fully wash out nonadsorbed substances. The adsorbed substances were eluted with 0.1-1 M NaCI linear gradient at a rate of 3 ml/min. By determining the antimicrobial spectrum of each fraction by the paper disc method (concentration: 10 ppm, pathogenic organisms: Botrytis cinerea), it was found that the active substance was contained in the final fraction eluted with about 0.68M NaCI.

The active fraction was lyophilised to concentrate it to 1/10 volume and was dialysed through a Visking tube to deionise it. The dialysed and deionised solution was lyophilised again to obtain thereby 10 mg of Botryticidin A as white powder.

EXAMPLE 2 Production of a Wettable Powder A mixture of 10 parts of Botryticidin A (concentrate before adsorption on CM-Sephadex C-25), 5 parts of sodium lauryl sulphate, 2 parts of sodium dinaphthyl-methane-disulphonate-formalin condensate and 83 parts of clay were blended and pulverised to yield 100 parts of a wettable powder.

EXAMPLE 3 Production of an Emulsifiable Liquid A mixture of 8 parts of Botryticidin A (concentrate before adsorption on CM-Sephadex C-25), 10 parts of ethylene glycol, 20 parts of dirnethylformamide, 10 parts of alkyl-dimethylbenzyl ammonium chloride and 52 parts of methanol were blended to obtain 100 parts of an emulsifiable liquid.

EXAMPLE 4 Preparation of a Dust A mixture of 0.2 part of Botryticidin A (concentrate before adsorption on CM-Sephadex C-25), 0.5 part of calcium stearate, 50 parts of talc and 9.3 parts of clay were blended and pulverised to obtain 100 parts of a dust.

EXAMPLE 5 Preparation of a Granule Product A mixture of 10 parts of Botryticidin A (concentrate before adsorption on CM-Sephadex C-25), 1 5 parts of starch, 72 parts of bentonite and 3 parts of sodium lauryl sulphate were blended and pulverised to obtain 100 parts of granules.

The effects of the agricultural and horticultural fungicide of the present invention on various plant diseases will now be described in detail by reference to the following further examples.

EXAMPLE 6 Test on Control of Grey Mould in Cucumbers A wettable powder prepared according to the method described in Example 2 was dissolved in water to a predetermined concentration and the solution obtained was sprayed onto cucumber seedlings (Sagami Hanjiro variety) obtained 1 5 days after seeding. The sprayed solution was air-dried.

A pathogenic fungus causing grey mould in cucumbers (Botrytis cinerea) was cultured in a potatoglucose-agar plate culture medium and was irradiated with Black Light Blue ray to induce spores. The spores were then suspended in an aqueous solution containing 10% of glucose and 1% of yeast extract.

After the suspension sprayed on the cucumber seedlings had dried, the cucumber seedlings were moved into an inoculation box. The suspension of spores was sprayed on the seedlings by a spray gun in an amount of 10 ml per 10 plants.

The plants thus inoculated were maintained at a temperature of 200C and at a relative humidity of 100% for 4 days and then the condition of the plants was observed.

The control value was calculated according to the following procedure.

Disease Index Disease Area Observed on Leaves 0 not observed 1 slightly observed 2 below 10% 3 below 20% 4 below 30% 5 below 40% 6 above 40%

the sum of disease index in treated area Control value (%) the sum = t x 1- 100 the sum of disease index in non-treated area The results obtained are given in Table I.

TABLE I Concentration of Fungicide in Sprayed Control Solution Value Chemical Tested (ppm) (%) Phytotoxicity Botryticidin A 100 100 not observed Botryticidin A 50 100 not observed Botrycidin A 25 99 not observed Botrycidin A 10 90 not observed Benlate 11 250 95 not observed Control 0 not observed NOTE: 1) Methyl-1 -(butylcarbamoyl)-L-benzimidazole carbamate.

EXAMPLE 7 Test on Control of Anthracnose in Cucumbers An emulsifiable liquid prepared according to the method described in Example 3 was diluted with water to a predetermined concentration and the dilute emulsion was sprayed on cucumber seedlings (Sagami Hanjiro variety) obtained 1 5 days after seeding. The sprayed emulsion was air-dried.

A pathogenic fungus causing anthracnose in cucumber (Colletotrichum lagenarium) was cultured in a potato-agar plate culture medium and the spores thus grown were suspended in water. The spore concentration of the suspension was adjusted to be such that about 200 spores were observed in the field of a microscope used at a magnification of 1 50 x.

After the sprayed emulsion had dried, the cucumber seedlings were transferred to an inoculation box. The spore suspension was sprayed on the seedlings using a spray gun in an amount of 10 ml per 10 plants. The seedlings thus inoculated were maintained in the inoculation box at a temperature of 20 C and a relative humidity of 100% for 24 hours and then moved into a greenhouse where they were subject to natural light for 4 days. Thereafter, the diseases in the plants were observed.

The control value was calculated according to the following formula:

the sum of disease spots in treated area Control value (%)= (%) sum = disease spot 1- x area x100 the sum of disease spots in non-treated area The results obtained are given in Table II.

TABLE II Concentration of Fungicide in Sprayed Control Emulsion Value Chemical Tested (ppm) (%) Phytotoxicity Botryticidin A 100 99 not observed Botryticidin A 50 95 not observed Botryticidin A 25 88 not observed Botryticidin A 10 79 not observed Daconil 2 1,250 95 not observed NOTE: 2) Tetrachloroisophthalonitrile.

EXAMPLE 8 Test on Control of Blast on Rice Ten stalks of rice (Jukkoku variety) were cultivated in a pot up to the four-leaves stage. A wettable powder prepared by the method described in Example 2 was dissolved in water to a predetermined concentration and the solution was sprayed on the plants by a spray gun in an amount of 50 ml per pot.

After the sprayed solution had dried, the rice plants were placed in an inoculation box and a suspension in water of spores of a pathogenic fungus causing blast on rice (Pyricularia oryzae) cultured in a rice hull culture medium (containing 3 g of rice hulls, 0.01 g of powdery yeast extract, 0.2 g of sucrose, 0.05 g of starch, and 5 ml of water) was uniformly sprayed onto the rice plants. After the pots had been kept in the inoculation box at a temperature of 250C and at a relative humidity of 100%, the plants were moved out of the inoculation box and kept under ambient conditions. When the disease was observed particularly in control plants 5 to 7 days after the inoculation, the number of disease spots per pot was counted. The control value was calculated according to the method as described in Test Example 2. The results obtained are given in Table Ill.

TABLE lil Concentration of Fungicide in Sprayed Control Solution Value Chemical Tested (ppm) (%) Phytotoxicity Botryticidin A 100 100 not observed Botryticidin A 50 100 not observed Botryticidin A 25 97 not observed Botryticidin A 10 95 not observed Blasticidin 53) 10 96 not observed NOTE: 3) Blasticidin-S-benzylaminobenzene-sulphonate.

EXAMPLE 9 Test on Control of Tomato Stem Rot A wettable powder prepared according to Example 2 was dissolved in water to a predetermined concentration and 50 ml of the solution were sprayed on tomato seedlings (Fukuju variety) which were at the seven to eight leaves stage after seeding. After the sprayed solution had air-dried, a piece of a fungal colony obtained by cutting a peripheral part of colony of Sclerstinia sclerotiorum with a cork borer (about 5 mm diameter) was patched and inoculated on a predetermined part of a leaf of each plant. The colony thus inoculated had been cultured in a conventional potato-glucose-agar plate culture medium. The tomato seedlings were then kept at a temperature of 250C and at a relative humidity higher than 95% for 3 days and the length of the disease spots was measured.The control value. was calculated according to the following formula:

/ the sum of lengths of disease spots in treated area Control value (%) = ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ x x 100 the sum of lengths of disease spots in non-treated areas The results obtained are given in Table IV.

TABLE IV Concentration of Antibiotic in Sprayed Control Solution Value Chemical Tested (ppm) (%) Phytotoxicity Botryticidin A 100 99 not observed Botryticidin A 50 90 not observed Botryticidin A 25 87 not observed Topsin M4 1000 85 not observed Control O NOTE: 4) [1,2-Phenylene bis (iminocarbonothioyl)j biscarbamic acid diethyl ester.

EXAMPLE 10 Test on the Control of Soft Rot of C;hinese Lettuce A wettable powder prepared according to Example 2 was dissolved in water to a predetermined concentration and 50 ml of the solution was sprayed on the seedlings of Chinese lettuce (Nozaki No. 2 variety) grown for 13 days after seeding. Three seedlings per area were used. After the sprayed solution had been air-dried, a suspension of cells of a pathogenic fungus which causes soft rot in vegetables (Erwinia carotovora) cultured in Suwa culture medium at 280C for 20 hours while shaking, was sprayed on the Chinese lettuce seedlings using a spray gun thereby to inoculate them.

After inoculation, the seedlings were maintained in an inoculation box at a temperature of 280C and at a relative humidity higher than 95% for 3 days and the degree of attack by the disease was then observed.

The control value was calculated according to the following procedure: Disease Index Disease Area on Leaves 0 not observed 1 very slightly observed 2 slightly observed 3 about 1/4 area 4 about 1/3 area 5 about 1/2 area 6 wholly attacked

Control value index in - ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ x 100 Control disease index in x 100 the sum of disease index in non-treated The results obtained are given in Table V.

TABLE V Concentration of Antibiotic in Sprayed Control Solution Value Chemical Tested (ppm) (%) Phytotoxicity Botryticidin A 100 81 not observed Botryticidin A 50 75 not observed Botryticidin A 25 69 not observed Agret51 100 68 not observed Control ~ 0 NOTE: 5) Streptomycin.

EXAMPLE 11 Test on Control of Powdery Mildew of Cucumber A wettable powder prepared according to Example 2 was dissolved in water to a predetermined concentration and 50 ml of the solution were sprayed on cucumber seedlings (Sagami Hanjiro variety) grown for 14 days after seeding. Three seedlings per area were used. The sprayed solution was dried. A pathogenic fungus causing powdery mildew in cucumber (Sphaerotheca fullginea) was inoculated on a grown cucumber leaf. A colony of the fungus was formed 11 days after inoculation. A suspension of spores obtained from the colony (the concentration of spores was 1 x 106 spores/ml) was sprayed onto the cucumber seedlings. The plants thus inoculated were moved into a greenhouse formed of vinyl film and kept at a temperature of 250 to 300 C, and the plants remained in the greenhouse for 11 days.

Thereafter, the number of disease spots on the leaves was counted.

The control value of the fungicide was calculated by the procedure of Example 7.

The results obtained are given in Table VI.

TABLE VI Concentration of Antibiotic in Sprayed Control Solution Value Chemical Tested (ppm) (%) Phytotoxicity Botryticidin A 100 90 not observed Botryticidin A 50 82 not observed Botryticidin A 25 79 not observed Morestan 61 60 85 not observed Control O NOTE: 6) Quinomethionate.

EXAMPLE 12 Test on Control of Angular Spot on Cucumber A wettable powder prepared according to Example 2 was dissolved in water to a predetermined concentration and 50 ml of the solution were sprayed on both sides of cucumber leaves (Suyo variety) at the two-leaves stage. After the sprayed solution had been air-dried, a suspension of spores of a pathogenic fungus causing angular spot on cucumber (Pseudomonas lachrymans) obtained by preculturing for 24 hours was sprayed on to the leaves of the cucumber seedlings. The seedlings thus inoculated were maintained in a greenhouse at a temperature of 250C for 2 days and then moved into another greenhouse subject to natural light. Thereafter, the degree of disease attack was observed.

The control value was calculated by the procedure of Example 10. The results obtained are given in Table VII.

TABLE VII Concentration of Antibiotic in Sprayed Control Solution Value Chemical Tested (ppm) (%) Phytotoxicity Botryticidin A 200 99 not observed Botryticidin A 100 85 not observed Botryticidin A 50 80 not observed Agret 50 70 not observed Control From the foregoing test results, it is apparent that Botryticidin A of the present invention is an effective fungicide, having very high control values with respect to various plant diseases while possessing no phytotoxicity. In other words, it is capable of wide and safe application to control many plant diseases to which plants are subject.

Claims (14)

1. The antibiotic, Botryticidin A characterised by the following physical, chemical and biological properties: (1) Elemental Analysis Values: C = 53.34%, H = 7.55%, N = 24.32%, 0 = 14.77%.

(2) Molecular Weight: 6,400.

(3) Specific Rotary Power: [a]2J: +1.10 (C = 0.02, in 0.8M-NaCI solution).

(4) Ultraviolet Absorption Spectrum determined in 0.9M NaCI solution as shown in Figure 1 of the accompanying drawings with maximum absorption observed at 207 nm (EI .005qó 0.3).

(5) Infrared Absorption Spectrum as shown in Figure 2 of the accompanying drawings with absorption bands being observed at wave numbers of 1630, 1210, 1160, 1110, 1090, 1040, 980, 730cm '.

(6) Solvent Solubility showing it to be soluble in water but only slightly soluble in common organic solvents including methanol, ethanol, butanol, ethyl acetate, ether, chloroform and benzene.

(7) Colour Reactions showing it to be positive to biuret reaction and ninhydrin reaction but negative to Folin reaction and Molisch reaction.

(8) Basic substance.

(9) Physical appearance: a white, amorphous powder.

(10) Thermostability as crude substance of Botryticidin A of as purified substance in a concentration of 200 ppm in NaCI solution above 0.6M shows it to be stable at a temperature ranging from room temperature to 1 000C, but having its antibiotic activity reduced when the crude Botryticidin A is subjected to deionisation.

(1 1 ) Amino Acid Composition: Amino Acid Number of Molecules Serine 11 Leucine 10 Glycine 9 Glutamic acid 7 (inclusive of Glutamine) Phenyl alanine 5 Alanine 4 Aspartic acid 3 (inclusive of Asparagine) Ornithine 3 Lysine 2 Histidine 2 Valine 2 Threonine 2 Arginine 1 Isoleucine 1 (12) Specific antifungal activity with respect to inter alia, Pyricularia oryzae, Rhizoctonia solani, Botrytis cinerea, Collectotrichum lagenarium, Alternaria kikuchiana, Glomerella cingulata and Ceratocystis finbriata.

2. A method for the production of Botryticidin A, which comprises culturing a Botryticidin Aproducing bacterium belonging to the genus Bacillus and separating and collecting the Botryticidin A from the culture medium.

3. A method according to claim 2, wherein the Botryticidin A-producing bacterium belonging to the genus Bacillus is Bacillus subtilis At 1316 (NRRL B-12231).

4. A method according to claim 3, wherein the Botryticidin A-producing bacterium belonging to the genus Bacillus is cultured in turn in a slant culture medium, a pre-seed culture medium, a seed culture medium and finally a main culture medium.

5. A method according to claim 4, wherein the separation of Botryticidin A from the main culture is carried out by the following steps: (a) centrifuging the culture medium containing Botryticidin A thereby to remove cells, (b) separating Botryticidin A from the supernatant by column chromatography, and (c) purifying the separated Botryticidin A by lyophilisation and dialysis.

6. A method of the production of pure Botryticidin A, substantially as described in the foregoing Example 1.

7. Botryticidin A, whenever produced by the method claimed in any one of claims 2 to 6.

8. An agricultural or horticultural fungicidal composition comprising Botryticidin A in association with a non-phytotoxic carrier therefor.

9. A composition according to claim 8, which is in the form of a settable powder containing Botryticidin A in an amount of 10 to 90% by weight.

10. A composition according to claim 8 which is in the form of an emulsion containing Botryticidin A in an amount of 10 to 90% by weight.

11. A composition according to claim 8, which is in the form of a dust containing Botryticidin A in an amount of 0.1 to 10% by weight.

12. A composition according to claim 8, which is in the form of granules containing Botryticidin A in an amount of 0.1 to 10% by weight.

1 3. An agricultural or horticultural fungicidal composition substantially as described in any one of the foregoing Examples 2 to 5.

14. A method of protecting an agricultural or horticultural crop against fungal attack which comprises applying to the crop a composition as claimed in any one of claims 8 to 13.