JP2673718B2 - Bacterial plant disease control method - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for suppressing disease of plants by microorganisms and cultivating healthy vegetables, and a novel microorganism belonging to the genus Bacillus used therefor.

Conventional technology Conventionally, many synthetic fungicides have been used to control diseases of agricultural and horticultural crops, but due to problems such as environmental impact and emergence of pathogens with drug resistance, crop pesticides have attracted attention. Has been done. That is, there is a method for controlling organisms that have an antagonistic action against pathogenic bacteria by directly adding to the plant body or upland soil to infect crops with disease fungi, suppressing the growth of disease fungi in upland soil or the disease activity. Being developed.

On the other hand, it has been found that antibiotics produced by microorganisms have a cure for various plant diseases. For example, certain strains belonging to the genus Bacillus are purmycin (4-ND), which is an antifungal antibiotic having a remarkable controlling effect against gray mold, sclerotium, powdery mildew, and aspergillosis of horticultural crops. It is known to produce -alanyl-2,4-diamino-2,4-dideoxyarabinose) (JP-A-54-1578).
96), and some other strains belonging to the genus Bacillus are known to produce the peptide antibiotic iturin A, which has a high control effect against gray mold, anthracnose, blast and blight. Has been. (JP-A-61-280095, Tetrahed
ron Letters No.30,3065-3068,1982).

However, the effects of these puromycins and iturin A are the effects when applied separately from the microorganisms, and no effective microorganisms are known for controlling the gray mold and powdery mildew by applying the microorganisms themselves to crops. .

Problems to be Solved by the Invention It would be extremely convenient if a microorganism producing an antibiotic could be directly applied to a crop without isolating the antibiotic produced from the above-mentioned microorganism from the microorganism. Development of microbial pesticides is desired.

The present invention broadly searches for microorganisms having the ability to produce primycin from the natural world, and utilizes microorganisms and microorganisms capable of suppressing diseases caused by Botrytis cinerea, powdery mildew, downy mildew, red rust, etc. It is an object of the present invention to provide a method for controlling plant diseases mainly caused by bacteria in vegetables.

Means for Solving the Problems The present invention belongs to the genus Bacillus separated from nature and has a novel ability to produce purmycin, a novel Bacillus subtilis spKB-1111 (Bacillus subtilis spKB-11).
11) Strain (Ministry of Industrial Science and Technology Article 1738) and Bacillus subtilis spKB-1122
It uses a strain (Microtechnology Research Institute, No. 1739).

Bacillus subtilis spKB-1111 strain (hereinafter simply referred to as KB
-1111) and Bacillus subtilis spKB-
The 1122 strain (hereinafter simply referred to as KB-1122 bacterium) produces an antibiotic such as purmycin when cultivated under the culturing conditions described below, and when this live bacterium is sprayed and produced on vegetables, gray mold, udon It has excellent effects against diseases, downy mildew, red rust, etc.

The strain KB- isolated by the present inventors from the soil of Teradomari, Niigata Prefecture
The 1111 bacterium has the following mycological properties.

Observation items KB-1111 a) Morphology (1) Shape and size Bacteria (rounded on both ends) (2) Polymorphic single (3) Motility and flagella (4) Presence or absence of spores Spore-shaped egg Circular spore formation center (5) Gram stain positive (6) No anti-acidity b) Growth status (1) Meat broth agar plate culture Colonies are grayish white or creamy, with a slight gloss, and are circular and have a diameter of 2 to 4 mm. .Ridge ridge type is concave, peripheral type is wavy and viscous (2) Sloped agar broth spreads and spreads on the surface of the medium, has a grayish white color with a slight gloss, is viscous, and has no diffusible pigment (3) broth liquid Medium On the first or second day, a pellicle is formed on the surface of the medium to cover the entire surface. The cells are grayish white without turbidity. (4) Potato slices Diffuse, wrinkled grayish white colonies (5) Meat broth gelatin puncture culture Liquefaction begins at 20 ° C and 30 ° C. The form is layered.

c) Physiological properties Test method 1) Reduction of nitrate with nitrate broth 2) Denitrification reaction Komagata's method None 3) MR negative 4) VP positive 5) No indole formation 6) Hydrogen sulfide formation TSI agar no acetic acid Method using lead test paper No meat juice No motility test medium 7) Use of citric acid Koser citrate medium Yes Christensen agar 8) Starch decomposition positive 9) Pigment formation No potato slice 10) Use of inorganic nitrogen source ammonium sulfate Yes Sodium nitrate No Glutamate soda Yes Casamino acid v · free Yes 11) Urease Christensen Urea medium Yes 12) Oxidase positive 13) Catalase positive 14) Casein degradation Casein 2% agar Yes 15) Litmus milk peptone and pigment reduction Yes 16) With LV agar 17) Without anaerobic growth of glucose broth 18) Range of growth (broth) Medium) Growth at 45 ℃ Yes No growth at 65 ℃ Yes Growth at pH5-9 Yes 7% NaCl growth 19) Lysozyme sensitivity 0.001% Glucose gravy 20) Acid production from sugar Glucose ◎ Sucrose ◎ Mannose ◎ Glycerin ◎ Sorbit ◎ Fructose ◎ Mannitol ◎ Xylose ◎ Arabinose ○ Starch △ Lactose ○ Maltose ◎ Inosit ○ Trehalose ○ Galactose × Raffinose △ ~ ○ 21) Sodium azide 0.02% growth No broth 22) Tyrosine decomposition No tyrosine agar On the other hand, the present inventor The strain KB-1122 isolated from the soil of Shinji-gun, Ibaraki prefecture has the following mycological properties.

Observation items KB-1111 a) Morphology (1) Shape and size Bacteria (rounded on both ends) (2) Polymorphic single (3) Motility and flagella (4) Presence or absence of spores Spore-shaped egg Circular spore formation center (5) Gram stain positive (6) No anti-acidity b) Growth condition (1) Meat broth agar plate culture Colonies are grayish white or cream-colored, non-glossy irregular circle size 2-6 mm in diameter. (2) Meat agar slope that spreads and spreads on the surface of the medium, and has a grayish white or cream color. There is no diffusible pigment. (3) Liquid medium for meat broth A pellicle is formed on the medium surface on the 1st or 2nd day. Cover all over. The cells are grayish white without turbidity. (4) Boiled potato slices Diffuse, wrinkled grayish white colonies (5) Meat broth gelatin puncture culture Liquefaction begins at 20 ° C and 30 ° C. Its shape is layered.

c) Physiological properties Test method 1) Reduction of nitrate with nitrate broth 2) Denitrification reaction Komagata's method None 3) MR negative 4) VP positive 5) No indole formation 6) Hydrogen sulfide formation TSI agar no acetic acid Method using lead test paper No gravy No motility test medium No 7) Use of citric acid Koser citrate medium Yes Christensen agar Yes 8) Starch decomposition positive 9) Pigment formation No jerk and potato slices 10) Inorganic nitrogen source Utilization test Ammonium sulfate Yes Sodium nitrate Yes Sodium glutamate Yes Casamino acid v ・ free Yes 11) Urease Christensen Urea medium Yes 12) Oxidase positive 13) Catalase positive 14) Casein degradation Casein 2% agar Yes 15) Litmus milk peptone and pigment reduction Yes 16) With LV agar Yes 17) No anaerobic growth of glucose broth 18) Range of growth (broth) Medium) Growth at 45 ℃ Yes No growth at 65 ℃ Yes Growth at pH5-9 Yes 7% NaCl growth 19) Lysozyme sensitivity 0.001% Glucose gravy 20) Acid production from sugar Glucose ◎ Sucrose ◎ Mannose ◎ Glycerin ◎ Sorbit ◎ Fructose ◎ Mannitol ○ Xylose ○ Arabinose ○ Starch ○ Lactose ○ Maltose ○ Inosit ○ Trehalose ○ Galactose △ Raffinose × 21) Sodium azide 0.02% growth No broth 22) Tyrosine decomposition Tyrosine agar From the above mycological properties, Bajay's Manual (Be
As a result of identification with reference to rgey's manual of systematic bacteriology, both strains were identified as species belonging to Bacillus subtilis.

Culturing conditions Cultivation of the above strains can be carried out according to a conventional method known in the field of fermentation science. As the medium, one containing an appropriate amount of carbon source and nitrogen source which can be assimilated by this strain and, if necessary, added an inorganic salt, a trace amount growth promoting substance and an antifoaming agent is used. Specifically, as a carbon source, glycose, fructose, maltose, galactose, ribose, saccharose, starch, molasses, sugars such as molasses sugar, glycerose, alcohols such as mannitol, fatty acids such as pyrivic acid, acetic acid and citric acid. , Glycine, glutamic acid,
One or two or more kinds may be appropriately selected and used from the general carbon sources such as amino acids such as glutamine, alanine and asparagine in consideration of the assimilation of the microorganism used.

As a nitrogen source, meat extract, peptone, yeast extract,
Dried yeast, soy hydrolyzate, soy flour, milk casein,
Organic nitrogen compounds such as casamino acids, various amino acids, corn starch-preca, etc., hydrolysates of animals, plants, microorganisms, ammonium salts such as ammonia, ammonium nitrate, ammonium sulfate, ammonium chloride, nitrates such as sodium nitrate, urea, inorganic nitrogen Considering the assimilation property of the microorganism to be used, one kind or two or more kinds may be appropriately selected and used from the compound.

Furthermore, as trace amounts of inorganic salts, magnesium, manganese, iron, calcium, potassium, etc., phosphates, hydrochlorides, sulfates, acetates and the like are appropriately added, or one or more species thereof are added, and vegetable oils and interfaces are added as necessary. An antifoaming agent such as an activator may be added.

The culturing is performed by selecting a culturing method suitable for the medium component to be used by a usual culturing method such as shaking culture, aeration and agitation culture, stationary culture, and continuous culture in a liquid medium containing the above-mentioned medium components.

The culture conditions may be appropriately selected depending on the type of medium and the culture method, and are not particularly limited as long as the strain can grow and produce puromycin, or if the strain can maintain the ability to grow and produce purmycin. Absent. Usually, it is preferable to adjust the pH at the start of cultivation to 6 to 8 and culture at a temperature condition of 25 to 35 ° C. The appropriate number of days for culture is usually 2 to 7 days.

After culturing the present strain as described above, the obtained culture itself, centrifugation from the culture, viable cells collected by a usual method such as aggregation separation, or freeze-dried viable cells, acetone Controlling downy mildew, red rust, powdery mildew, gray mold and the like by applying to the crop dried cells that have been dried by a method such as drying or those coated with a coating agent be able to.

Incidentally, as the above-mentioned coating agent for bacterial cells, a coating agent used for coating plant seeds, for example, methyl cellulose, gum arabic, alginate, polyurethane prepolymer, natural or synthetic polymer such as polyvinyl acetate homopolymer, calcium peroxide. , Calcium carbonate, calcined gypsum, glass cotton, etc. can be used.

It can be known from the following that KB-1111 and KB-1122 according to the present invention produce puromycin.

That is, the supernatant obtained by removing the cells from each culture solution of KB-1111 bacteria and KB-1122 bacteria was adjusted to pH 3.0 and treated with a cation exchange resin Amberlite IRC-50 (manufactured by Organo), The adsorbed component was eluted with 0.5N-ammonia water,
After neutralization, adsorption treatment with Amberlite CG-50 (manufactured by Organo), elution of the adsorbed component with 0.5N ammonia water, neutralization, and adsorption treatment with CM-Sephadex (manufactured by Pharmacia) Was eluted with 0.6 mol of saline solution,
Further, it was treated with Sephadex LH-60 (manufactured by Pharmacia), and the adsorbed component was eluted with water and concentrated to obtain a white crystal. As a result of the analysis, this crystal was puromycin.

 Hereinafter, the present invention will be described specifically with reference to examples.

Example 1 (Example of production of viable cells) Meat extract 3 g, peptone 10 g, and sodium chloride 5 g were dissolved in purified water 1000 ml, pH was adjusted to 7.0 to 7.3, and autoclave sterilized, KB-1111 bacteria and KB-1122 bacteria respectively. Inoculate separately and culture at 30 ° C for 1 day. 1-5% of this preculture
Inoculate the following main culture medium. The main culture is shaking culture at 30 ° C. for 5 days. After culturing, the bacterial cells are collected and freeze-dried.

Main culture medium composition Yeast extract 0.5 g, hydrogen phosphate-potassium 2 g, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate 0.5 g, ferrous sulfate heptahydrate 2 mg, Pharmamedea 20 g, lactose 20 g, tap water 1000 ml The pH is adjusted to 7.2 and sterilized by autoclaving at 120 ° C for 20 minutes.

Example 2 The freeze-dried bacterial cells obtained according to Example 1 were subjected to a gray mold control test using isogen according to the following method.

Isogen Cotyledon Gray mold test method Drug solution (KB-1111 or KB-11)
22 Dry cells (10 to 15 ml / one isogen) are evenly sprayed onto isogen cotyledons (postemergence drug 10 days). After the air-drying of the drug solution, the colony tip of Botrytis cinerea, which had been grown on potato-saccharose agar medium, was punched out with a cork borer (agar piece), 2 per leaf,
Inoculate a total of 4 on the cotyledons. Then add the isogen pot 20
It is kept at high temperature and high humidity, and the degree of illness is investigated after 3 to 4 days.
The degree of disease onset is expressed as a percentage of the lesion area formed on the isogen leaves relative to the lesion area formed on the untreated plot of the isogen leaves.

The results are shown in Table 1. In addition, the results using commercially available control agents are also shown for comparison.

In addition, the results of using the commercially available control agents are also shown in Tables 2 to 4 below for comparison.

Example 3 Cucumber bean and disease control effect test KB-on a cucumber leaf (cultivar; Sagamihanjiro, 1 sowing / pot, 3 pots / treatment group used) when the second true leaf was cultivated in a biscuit pot with a diameter of 10 cm 1111 and KB-1122 cultured cells were diluted and suspended in water to 0.7% (dry cell weight%) and sprayed in an amount of 5 ml per pot. After air-drying the sprayed leaves, spray inoculate the suspension of cucumber beetle and diseased spores collected from the diseased leaves and incubate them at 20-22 ° C under high humidity for 24 hours.
It was kept for a time and then left in the greenhouse. On the 5th to 7th day after the inoculation, the lesion area ratio of cucumber and disease was investigated, and the control value was calculated by the following formula.

The results are shown in Table 2.

Example 4 Wheat Powdery Mildew Control Effect Test For the second leaf stage seedling wheat (cultivar; Norin No. 64, 16 plants / pot, 3 pots / treatment group used) cultivated in a biscuit pot with a diameter of 10 cm
KB-1111 and KB-1122 cultured bacterial cells were diluted and suspended in 0.7% (dry cell weight%) with water and sprayed at 5 ml per pot. After air-drying the sprayed leaves, a suspension of Komugi powdery mildew fungus spores collected from the diseased leaves was spray-collected, kept at a high temperature of 20 to 24 ° C. for 24 hours, and then left in a greenhouse. 9 to 11 days after the inoculation, the lesion area ratio of wheat powdery mildew was investigated, and the control value was calculated by the following formula.

The results are shown in Table 2.

Example 5 Cucumber Powdery Mildew Control Effect Test For cucumber (cultivar; Sagamihanjiro, 1 / pot, 3 pots / treatment group used) when the second true leaf was cultivated in a biscuit pot with a diameter of 10 cm
KB-1111 and KB-1122 cultured bacterial cells were diluted and suspended in 0.7% (dry cell weight%) with water and sprayed at 5 ml per pot. After air-drying the sprayed leaves, spores were sprinkled on the sick leaves to inoculate them, and the spores were infected in a glass greenhouse. The lesion area ratio of cucumber powdery mildew was investigated 9 to 11 days after the inoculation, and the control value was calculated by the following formula.

The results are shown in Table 2.

Example 6 Wheat leaf rust control effect test KB-1111, KB-11 on seedling wheat (variety: Norin 64, 16 / pot) seedlings at the time of the second true leaf cultivated using a clay pot with a diameter of 10 cm.
22 cultured cells were diluted and suspended in water to 0.7% (dry cell weight%) and sprayed at a rate of 5 ml / pot. The sprayed leaves were air-dried, spray-inoculated with a suspension of wheat rust spores collected from the diseased leaves, and kept at 20-23 ° C high temperature for 24 hours. Then, it was left in a glass greenhouse, 7 to 10 days after the inoculation, the lesion area ratio of wheat leaf rust was investigated, and the control value was calculated by the following formula.

The results are shown in Table 2.

Example 7 Lettuce gray mold control effect test KB-1111, KB-on leaves of lettuce (variety: King Crown) cultivated in a greenhouse (15 to 25 ° C.) for about 5 weeks after sowing in a clay pot with a diameter of 10 cm 1122 cultured bacterial cells were diluted to a predetermined concentration with water and suspended, and 5 ml was sprayed per pot. After air-drying the sprayed leaves, the fungal medium of gray mold fungus is preliminarily used (5 g of bran, 1 g of chaff, 5 m of water.
l), cultivated at 20 ℃ for 8 days, inoculate the lump of mycelium that has been sewn through a 16-mesh sieve, and inoculate directly from the top of the lettuce.
It was kept under high humidity conditions at 22 ° C. On the 5th day after the inoculation, the lesion area ratio of gray mold of lettuce was investigated, and the control value was calculated by the following formula.

The results are shown in Table 3.

Example 8 Gray mold control effect test of strawberries Strawberry (variety: Onamine) fruits are arranged using a strawberry pack, and KB-1111 and KB-1122 cultured cells are 0.7% (dry cell weight%). The suspension diluted with water was sprayed at a rate of 5 ml per pack. After air-drying, 1 spore of Botrytis cinerea was cultivated for 14 days at 20 ° C using sugar-added potato decoction agar in advance.
An equivalent amount of 1/4 petri dish was applied per pack and kept under high humidity conditions at 20-22 ° C. On the 4th day after the inoculation, the lesion area ratio of gray mold of strawberry was investigated, and the control value was calculated by the following formula.

Table 4 shows the results.

Claims (3)

(57) [Claims] 1. A Bacillus subtilis spKB-1111 (Baci
llus subtilis spKB-1111) strain
No.) and / or Bacillus subtilis spKB-1122 (Ba
cillus subtilis spKB-1122) strain
No.) is sprayed and grown on vegetables to control plant diseases against bacteria. 2. Bacillus subtilis spKB-1111 (Bacillus subtilis sp) having plant disease control activity against bacteria.
KB-1111) strain (Ministry of Fine Arts, Article 1738). 3. A Bacillus subtilis sp KB-1122 (Bacillus subtilis sp) having a plant disease control activity against bacteria.
KB-1122) strain (Ministry of Mechanical Engineering, Article 1739).