KR20060115765A - Pland disease controlling composition and microorganism - Google Patents

Abstract

[PROBLEMS] To provide a novel microorganism belonging to the genus Bacillus; a plant disease controlling composition capable of controlling diseases on various agricultural and horticultural corps even under low-temperature conditions frequently allowing the occurrence of plant diseases (in particular botrytis rot, sclerotal disease, powdery mildew, anthracnose, ring spot disease, blue mold disease, green mold disease, soft rot, ulcer, etc.); a method of controlling plant diseases by using the plant disease controlling composition; and a method of selecting the above-described microorganism. [MEANS FOR SOLVING PROBLEMS] A plant disease controlling composition containing a novel microorganism belonging to the genus Bacillus. As specific examples of this microorganism, Bacillus subtilis 4-5-1-1 strain No.30 and Bacillus sp. 4-5-21 0306 strain can be cited. A method of effectively controlling botrytis rot, sclerotal disease, powdery mildew, anthracnose, ring spot disease, blue mold disease, green mold disease, soft rot and ulcer by treating a plant and/or soil with the plant disease controlling composition as described above. A strain mainly derived from the above- described microorganism with a slight change in its characteristics. A method of selecting the above-described microorganism.

Description

Plant disease control composition and microorganism {PLAND DISEASE CONTROLLING COMPOSITION AND MICROORGANISM}

The present invention provides a variety of agricultural and horticultural crops, such as botrytis rot, sclerotal disease, powdery mildew, anthracnose, ring spot disease, and blue mold disease. A new family belonging to the genus Bacillus, which has the ability to control in the low temperature region of the plant disease occurrence, for plant diseases such as yeast mold, green mold disease, soft rot, and ulcer. The present invention relates to a plant disease control composition having a microorganism, the novel microorganism, and a plant disease control method using the plant disease control composition.

Conventionally, benzimidazole type fungicides and thiophanate type fungicides have excellent control effects against various pathogens parasitic in agrohorticultural crops, and since 1970, they have been widely used as agrohorticultural fungicides to greatly increase crop yield. Has contributed.

On the other hand, biological pesticides are known as a means of replacing or using conventional synthetic fungicides with very low environmental pollution, harmonizing with ecosystems, and having excellent control effects. As microorganisms that have been used for controlling diseases of agro-horticultural crops, in particular, gray fungus, microorganisms belonging to the genus Tricoderma, Gliocladium, and Bacillus, and these microorganisms to date Many agricultural and horticultural fungicide compositions containing these have been researched and developed.

For example, regarding the bacteria belonging to the genus Bacillus, Japanese Patent Laid-Open No. 63-273470 discloses Bacillus subtilis JB3 strain (NCIB12375), Bacillus subtilis JB3.6 strain (NCIBB12376). Culture of the above strains by antimicrobial substances obtained from Bacillus subtilis R1 strain (NCIB12616), or mutant strains thereof, which inhibit plant diseases, animal and human microbial contamination, and also suppress general microbial contamination. Attempts have been made to control diseases of various agricultural horticultural crops using water (see Patent Document 1, for example). In addition, the antibacterial material obtained from the Bacillus subtilis JB3 strain (NCIB12375), Bacillus subtilus JB3.6 strain (NCIBB12376), Bacillus subtilus R1 strain (NCIB12616), or mutant strains thereof is isolated, Attempts have been made to use this for disease control of various agricultural horticultural crops (see Patent Document 2, for example). In addition, Bacillus amyloliquefaciens is known as a microorganism for controlling anthracnose and white root rot of mulberry trees (for example, Patent Document 3, Non-Patent Document 1, and Non-Patent Document 2). Reference). Moreover, the impression hydration agent which is an apo formulation of Bacillus subtilis QST-713 strain obtained the pesticide registration in May 2003, and it is put on the market (for example, refer nonpatent literature 3).

Patent Document 1: Japanese Unexamined Patent Publication No. 63-273470

Patent Document 2: Japanese Patent Application Laid-Open No. 2-22299

Patent Document 3: Japanese Patent Application Laid-Open No. 11-246324

Non Patent Literature 1: Plant Control Vol. 56, No. 8 (2002)

Non Patent Literature 2: Phytopathology Vol. 91 No. 2, 2001

Non-Patent Document 3: Biocontrol Vol. 7 No. 1, 2003

Disclosure of the Invention

Problems to be Solved by the Invention

However, in the method for controlling pests of agrohorticultural crops applied to bacteria belonging to the genus Bacillus, the difference between the growth temperature of plant pathogens and the growth temperature of the controlling bacterium is large, and in both low temperature areas where various agronomic crop diseases occur. Control was not necessarily enough. For example, gray fungal disease of various agro-horticultural crops is very active at low temperatures around 15 ° C, and particularly in bacteria belonging to the genus Bacillus having growth temperature at 20 ° C to 45 ° C, especially when the temperature is low. A sufficient control effect could never be obtained (for example, see nonpatent literature 3).

The present invention has been made in view of such circumstances, and the problem of the present invention is that even in a low temperature region such as 10 ° C. to 15 ° C. with respect to diseases of plants such as various horticultural crops, in particular to plant diseases such as gray mold disease, etc. Microorganisms belonging to the genus Bacillus that have a consistently good control ability, a plant disease control composition containing the microorganisms, a method for controlling plant diseases using the same to treat plants and / or soil, and mainly to the microorganisms The present invention provides a strain derived from the above-described strain, and a method for selecting the microorganism.

Means to solve the problem

MEANS TO SOLVE THE PROBLEM In order to solve the said subject, the present inventors isolate the candidate microorganism which has plant disease control ability from the tea leaves collected from each package of Nippon Soda Co., Ltd. Haibara Field Research Center. The leaf surface of the tea leaves collected from each package was washed in a small amount of sterile water, and the washed water was spread on a standard agar medium (manufactured by Nissei Pharmaceuticals Co., Ltd.) to collect colonies of the grown microorganisms, and again to become single colonies. Separation and acquisition were made. Among these microorganisms, strains having a control effect against gray mold (Botrytis cinerea) were selected at room temperature (20 ° C). Regarding the selected strains, strains viable at low temperature (10 ° C) were selected again, and two strains of microorganisms having a control effect against gray mold disease were found again at low temperature (14 ° C). These two strains are expected to be bacteria of the genus Bacillus from the form of colonies, and their forms and mycological properties were as follows.

It was a Gram-positive bacterium that was catarrase positive, produced aerobic acid, and formed Bacillus-like apo.

As a result of systematic analysis based on 16 SrDNA sequences of about 1100 bp, the 4-5-1-1 strain No.30 was Bacillus subtilis and Bacillus amyloliquefaciens. (Bacillus amyloliquefaciens), Bacillus velesensis (99.906%), and Bacillus licheniformis (99.719%). In addition, as a result of using API 50 CH of the Api manual kit (bio Merieux Industry), the 4-5-1-1 strain No. 30 has a homology of 44.7% to Bacillus subtilis and Bacillus riche. The homology to Niformis (Bacillus licheniformis) was 35.1%. In addition, as a result of examining the anaerobic growth, the use of propionate, and the growth at 55 ° C, all were negative. As a result of investigation of the availability of the carbon compound, 4-5-1-1 strain No.30 was identified as Glycerol, L-Arabinose, Ribose, D-Xylose, D-Glucose, D-Fructose, D-Mannose, Inositol, and Mannitol. Sorbitol, α-Methyl-D-glucoside, Amygdaline, Arbutin, Esculin, Salicin, Cellobiose, Maltose, Lactose, Melibiose, Sucrose, Trehalose, D-Raffinose, Starch, Glycogen, β-Gentiobiose are available, Erythritol, D -Arabinose, L-Xylose, Adonitol, β-Methyl-D-xyloside, Galactose, L-Sorbose, Rhamnose, Dulcitol, α-Methyl-D-mannoside, N-Acethyl-glucosamine, Inuline, Melezitose, Xylitol, D-Turanose , D-Lyxose, D-Tagatose, D-Fucose, L-Fucose, D-Arabitol, L-Arabitol Gluconate, 2-ceto-gluconate and 5-ceto-gluconate were not available.

On the other hand, in the 4-5-21 0306 strain, the homology search was performed based on the gene analysis based on the about 700 bp sequence of 16SrDNA, and the homology agreement rate with the Bacillus subtilis was 99.0%. In addition, as a result of using API 50 CH of the bio manual kit (bio Merieux Industry), the 4-5-21 0306 strain had a homology of 87.4% with Bacillus subtilis. In addition, as a result of examining the availability of the carbon compound, 4-5-21 0306 strain Glycerol, L-Arabinose, Ribose, D-Glucose, D-Fructose, D-Mannose, Inositol, Mannitol, Sorbitol, α-Methyl-D- Glucoside, Amygdaline, Arbutin, Esculin, Salicin, Cellobiose, Maltose, Lactose, Sucrose, Trehalose, D-Raffinose, Starch, Glycogen are available, Erythritol, D-Arabinose, D-Xylose, L-Xylose, Adonitol, β- Methyl-D-xyloside, Galactose, L-Sorbose, Rhamnose, Dulcitol, α-Methyl-D-mannoside, N-Acethyl-glucosamine, Melibiose, Inuline, Melezitose, Xylitol, β-Gentiobiose, D-Turanose, D-Lyxose, D-Tagatose, D-Fucose, L-Fucose, D-Arabitol, L-Arabitol Gluconate, 2-ceto-gluconate and 5-ceto-gluconate were not available.

In total, the 4-5-1-1 strain No. 30 was judged to be Bacillus subtilis. In addition, 4-5-21 0306 strain was determined to be Bacillus sp., A recent species of Bacillus subtilis. These strains were assigned to the Patent Biological Deposition Center of the Industrial Technology Research Institute (Individual Administration) (1, 6 Daimon, 1jome, Higashi, Tsukuba City, Ibaragi Prefecture, Japan) on February 10, 2004, respectively, accession number FERM BP-10244 and accession number FERM. Deposited as BP-10245. In addition to the gray mold, the bacterium has excellent control activities against a wide range of diseases such as fungal nucleus disease, powdery mildew, anthrax disease, leap disease, blue mold disease, yeast fungus disease, soft disease, ulcer disease, and the like.

That is, the present invention

First, a commercially available Bacillus spp. Is a plant pest control composition comprising microorganisms belonging to Bacillus sp. Having high disease control ability under low temperature conditions that do not exhibit a sufficient effect,

2nd, low temperature conditions are 10-15 degreeC, It is a plant disease control composition of 1 characterized by the above-mentioned,

3, the plant disease control composition according to 1 or 2, wherein the plant disease is at least one of gray mold disease, fungal disease, powdery mildew, anthrax, leap disease, blue mold disease, yeast mold disease, soft disease, and ulcer disease. ego,

The microorganism which belongs to 4th, Bacillus sp. Is Bacillus subtilis 4-5-1-1 strain No.30, The plant disease control in any one of 1-3 characterized by the above-mentioned. Is a composition,

The microorganism which belongs to 5th, Bacillus sp., Is a plant disease control composition in any one of 1 to 3 characterized by the owner of Bacillus sp. 4-5-21 0306,

6, the microorganism belonging to the genus Bacillus sp. Is a strain mainly derived from Bacillus subtilis 4-5-1-1 strain No. 30, and changed only a few of its characteristics. It is a plant disease control composition in any one of 1-3 characterized by the above-mentioned,

Microorganisms belonging to the seventh and Bacillus sp. (Bacillus sp.) Is a strain mainly derived from Bacillus sp. 4-5-21 0306 strain, which has changed only a few of its characteristics. It is a plant disease control composition in any one of 3,

It is a plant disease control method characterized by treating a plant and / or soil using the plant disease control agent composition in any one of 8th, 1-7.

9th, Bacillus subtilis 4-5-1-1 strain No. 30,

10, Bacillus sp. 4-5-21 0306 week,

11th, Bacillus subtilis (Bacillus subtilis) is a strain mainly derived from No. 30 of 4-5-1-1 strain, and changed only a few of its characteristics,

Twelfth, Bacillus sp. 4-5-21 strains mainly derived from 0306, the characteristics of which have changed only a few of them,

The method for selecting microorganisms belonging to the genus Bacillus sp., Which has high activity at low temperature according to item 13, which consists of successive steps of a to d of the thirteenth.

a. Process of collecting bacteria from strain isolate

b. Process for selecting strains having a control effect against gray mold (Botrytis cinerea) at room temperature (18 ~ 25 ℃)

c. Process for selecting strains that can grow at low temperature (8-12 ℃)

d. Reselecting a strain having a control effect against gray mold (Botrytis cinerea) at low temperature (13-15 ℃).

Effects of the Invention

The plant disease control composition of the present invention contains a microorganism having a high disease control ability under a low temperature condition in which a commercially available Bacillus agent does not exhibit a sufficient effect, and thus high disease under a low temperature condition in which a commercially available Bacillus agent does not exhibit a sufficient effect. The control ability can be exhibited, the disease control ability can be maintained over a long period of time, and various kinds of disease can be controlled at once. According to the method for controlling plant diseases of the present invention using such a plant disease control composition, plant diseases, in particular, gray mold, are sprayed on plants such as various agricultural horticultural crops or soil by spraying the plant disease control composition. The disease can be effectively controlled. In addition, according to the selection method of the present invention, microorganisms having high disease control ability can be efficiently selected under low temperature conditions in which commercially available Bacillus sp. Preparations do not exhibit sufficient effects.

To practice the invention  Best form for

As a plant disease control composition of the present invention, it can be separated from tea leaves, has a control effect against gray mold (Botrytis cinerea), and also has a good control ability in all disease-inducing temperature range, especially low temperature region Bacillus Microorganism belonging to the genus Bacillus sp., Bacillus subtilis 4-5-1-1 strain No.30 (FERM BP-10244), Bacillus sp. 4-5-21 strain 0306 Although it does not restrict | limit especially if it is a plant disease control composition containing at least 1 sort (s) chosen from (FERM BP-10245), The microorganism belonging to the genus Bacillus sp. Is S. sclerotiorum Against anthrax (C. lagenarium), P. longiseta, yeast fungus (P. digitatum), blue fungus (P. italicum), soft disease (E. carotovora), ulcer disease (X. campestris) It is preferable to have a control effect.

The plant disease control composition of the present invention can be used particularly effectively against gray mold disease, fungal nucleus disease, powdery mildew, anthrax disease, leap disease, blue mold disease, yeast mold disease, soft disease, ulcer disease and the like.

In the production of the plant disease control composition of the present invention, cells which have grown microorganisms belonging to the genus Bacillus sp. By a known means such as solid culture or liquid culture can be used. As a method for propagating such microorganisms, any method may be used regardless of the type of medium, the culture conditions, or the like, as long as the cells are grown. For example, in the case of solid culture, the standard agar medium, the agar medium, and the potato deck In the case of the liquid culture, the stationary culture at 25 to 35 ° C. in the Straw agar medium and the like, shaking and stirring at 25 to 35 ° C. in various liquid mediums from which the agar is removed from the agar medium, and the like can be given. The cells of the present invention refer to all forms that microorganisms of the genus Bacillus, such as feeder cells or spores, can usually take. In addition, as the use form of the cells, any form such as suspensions or culture liquids or concentrates thereof, pastes, dried products, dilutions, etc. besides the cells itself can be applied.

The concentration of the bacterium belonging to the genus Bacillus sp. In the disease control composition of the present invention is not particularly limited, but when diluted to 1000-2000 times, it is converted to the cell concentration and converted into a cell concentration of 1 × 10 ^{11 to} 1 × 10. ² cfu / ml, Preferably the range of 1x10 <^9> -1 * 10 <^4> cfu / ml can be illustrated preferably. In addition, the pest control composition of the present invention may be formulated with a carrier, a surfactant, a dispersant, an adjuvant, and the like, which are commonly used, and in a form that can be taken by conventional pesticides, for example, powders, hydrating agents, etc. , Oils, floors, granules and the like can be employed.

Examples of the agrochemical preparations of the present invention include alumina, such as inorganic salts such as calcium carbonate, potassium chloride, sodium sulfate, calcium sulfate, and ammonium sulfate, organic acids such as citric acid, malic acid, stearic acid, sugars such as salts, lactose and sucrose, and the like. Solid carriers such as powder, silica gel, zeolite, hydroxyapatite, zirconium phosphate, titanium phosphate, titanium oxide, zinc oxide, hydrotalcite, kaolinite, montmorillonite, talc, clay, diatomaceous earth, bentonite, white carbon, kaolin, vermiculite Can be mentioned. Moreover, antioxidant, a coloring agent, a lubricating agent, a ultraviolet absorber, an antistatic agent, etc. can be added as needed. And as an adjuvant, carboxymethylcellulose, polyethyleneglycol, gum arabic, starch, etc. can be added, for example.

Surface active agent and the dispersing agent is not limited particularly as long as it can be used in conventional agricultural chemical preparations, specifically, a non-ionic surfactant is a sorbitan fatty acid ester (C _{12 ~18),} POE sorbitan fatty acid ester (C _12-18), ester-type surfactants such as sucrose fatty acid esters of sugar, POE fatty acid esters (C _12-18), POE resin acids esters, POE fatty acid diester (C _12-18) fatty acid ester such as a surfactant, POE alkyl ether (C _{12 ~18)} alcohol surfactants, POE alkyl, such as (C _{8 ~12)} phenyl ether, POE dialkyl (C _{8 ~12)} ether, POE alkyl (C _{8 ~12)} phenyl ether formalin condensate alkyl phenol surfactants such as water, and a polyoxyethylene polyoxypropylene block polymer, alkyl (C _{12 ~18)} of polyoxyethylene and polyoxypropylene and polyoxyethylene poly-propylene oxide block polymers such as ether Polypropylene block polymer type surfactant, POE alkylamine (C _{12 ~18),} POE fatty acid amide (C _{12 ~18)} a bisphenol-type surfactants such as alkyl amine surfactants, POE fatty acid bisphenyl ether, such as, POA benzyl phenyl ( Or polyaromatic cyclic surfactants such as phenylphenyl) ether and POA styrylphenyl (or phenylphenyl) ether, silicon-based fluorine-based surfactants such as POE ether and ester-type silicon and fluorine-based surfactants, fluorine-based castor oil, and POE-cured castor a plant type surfactants, anionic surfactants such as alkyl sulfates are free _(~18 C _12, Na, NH _4, alkanolamine), POE alkyl ether sulfates _{(C 12 ~18, Na, NH} 4, alkanes olamine), POE alkylphenyl ether sulfate (C _{12 ~18,} NH _4, alkanolamine, Ca), POE benzyl (or styryl) phenyl (or phenylphenyl) ether sulfate (Na, NH _4, alkanol Amines), polyoxyethylene, polyoxypropylene block Polymer sulfate (Na, NH _4, alkanolamine), such as a sulfate surfactant, a paraffin (alkane) sulfonate (C _{12 ~22,} Na, Ca, alkanolamine), AOS (C _{14 ~16,} Na , alkanolamine), dialkyl sulfosuccinate (C _{8 ~12,} Na, Ca, Mg), alkyl benzene sulfonate (C _12, Na, Ca, Mg, NH _4, alkylamine, alkanol, amine, cyclohexylamine), mono- or di-alkyl (C _{3 ~6)} naphthalene sulfonate (Na, NH _4, alkanolamine, Ca, Mg), naphthalene sulfonate-formalin condensate (Na, NH _4), alkyl (C _{8 to 12)} diphenylether sulfonate (Na, NH _4), lignin sulfonate (Na, Ca), POE alkyl (C _{8 to 12)} phenyl ether sulfonate (Na), POE alkyl (C _{12 ~18)} ether sulfosuccinic acid half esters (Na), such as a sulfonate surfactant, carboxylic acid type fatty acid salt (C _{12 ~18,} Na, K, NH _4, alkanolamine), N- methyl-fatty acid live Cauchy carbonate (C _{12 ~ 18} , Na), resinate (Na, K), etc. POE alkyl (C _{12 ~18)} ether phosphate (Na, alkanolamine), POE mono- or dialkyl (C _{8 ~12)} ether phosphate (Na, alkanolamine), POE benzyl (or styryl) phenyl screen (or phenyl phenyl) ether phosphate (Na, alkanolamine), polyoxyethylene and polyoxypropylene block polymers (Na, alkanol amine), phosphatidyl choline and phosphatidyl ethanol imine (lecithin), alkyl (C _{8 ~12)} phosphates, such as phosphate-type surfactants, cationic surfactants include alkyl trimethyl ammonium chloride (C _{12 ~18),} and methyl polyoxyethylene alkyl ammonium chloride and (C _{12 ~18),} alkyl and N- methyl pyridium bromide (C _{12 ~18)} , mono- or di-alkyl (C _12~18) methylated ammonium chloride, alkyl (C _{12 ~18)} pentamethyl propylene diamine di-ammonium-type surfactant include alkyl dimethyl benzalkonium chloride, such as benzalkonium chloride (C _{12 ~18),} benzethonium chloride la De (octyl phenoxy ethoxy ethyl dimethyl benzyl ammonium chloride) as a benzalkonium-type surfactants, amphoteric surfactants such as dialkyl (C _{8 ~12)} diamino ethyl betaines, alkyl (C _{12 ~18)} dimethylbenzyl beta the like can be mentioned the beta doll surfactants, such as dialkyl (C _{8 ~12)} diamino ethyl glycine and alkyl glycidyl new surface active agents such as (C _{12 ~18)} dimethyl benzyl glycine. These can be used individually by 1 type or in mixture of 2 or more types.

In addition, it can be in the form of powder, hydrated agent, emulsion, floorable agent, granule, etc., using methods used in the preparation of conventional chemical pesticides and biological pesticides and materials such as carriers, surfactants, and dispersants. .

The control method of the plant disease of the present invention is not particularly limited as long as it is a method of controlling the plant disease using the plant disease control composition of the present invention, and the plant disease control composition of the present invention is similar to conventional chemical pesticides. The method of spraying to plants or soil, such as various agricultural and horticultural crops, is mentioned. Moreover, as described in Unexamined-Japanese-Patent No. 2001-302407, the preparation can also be installed in the vicinity of the tuyere of the blower which blows in a facility, and an agricultural chemical can be sprayed with the air blown out from the tuyere. In the spraying process, can be used to dilute the plant disease control agent composition of the present invention in water and the like in a suitable amount, as salporyang is, in terms of cell density belonging to Bacillus (Bacillus sp.), Typically 1 × 10 ¹¹ ~ 1 × 10 ² cfu / ml, preferably 1 × 10 ^{9 to} 1 × 10 ⁴ cfu / ml.

Under the low temperature conditions in which the commercially available Bacillus genus preparation of this invention does not show sufficient effect, it can show 10-15 degreeC specifically. In addition, a strain mainly derived from a specific strain of the present invention and altering only some of its characteristics includes a method of selecting a naturally occurring or artificially generated variant, a method of obtaining a transformed strain by introducing a separate gene, and cell fusion. The strain obtained by the method etc. which obtain the strain obtained by the asymmetric fusion of the middle can be shown.

The selection method of Bacillus sp. Which has high activity at low temperature which consists of each successive process of a to d of the present invention can specifically represent the following method. As a step of collecting the cells from the strain isolate of step a, a conventionally known method can be applied. The normal temperature of step b indicates the temperature at which the target plant pathogen can normally grow, and specifically 16 to 25 ° C, preferably 20 ° C. The low temperature of the process c shows the vicinity of the minimum temperature at which the propagation of the target plant pathogen stops, and specifically, it is 8-12 degreeC, Preferably it is 10 degreeC. The low temperature of the step d represents an intermediate temperature between the normal temperature and the low temperature of the step c, and specifically 13 to 15 ° C, preferably 14 ° C.

Hereinafter, although the plant disease control composition of this invention and the plant disease control method using this are demonstrated concretely by an Example, this invention is not limited to a following example at all, The range which does not deviate from the meaning of this invention In, Bacillus sp. Can be freely changed the type of bacteria, the composition ratio of the preparation, the formulation, and the like. In the following examples, two strains of FERM BP-10244 and FERM BP-10245 were used as Bacillus sp.

Example 1

Selection of Bacillus subtilis

Candidate microorganisms having plant disease control ability were isolated from the tea leaves collected at each package of Nippon Soda Co., Ltd. Haibara Field Research Center. The leaf surface of the tea leaves collected from each package was washed in a small amount of sterile water, and the washed water was spread on a standard agar medium (manufactured by Nissei Pharmaceuticals Co., Ltd.) to collect colonies of the grown microorganisms, and again to become single colonies. Separation and acquisition were made. Among these microorganisms, strains having a control effect against gray mold (Botrytis cinerea) were selected at room temperature (20 ° C). Regarding the selected strain, Bacillus subtilis 4-5-1-1 which reselects the viable strain at low temperature (10 ° C), and has control effect against gray mold disease at low temperature (14 ° C) again. Week No. 30, and Bacillus sp. 4-5-21 0306 week were found.

Example 2

Preparation of Bacillus subtilis Cell Suspension

150 ml of standard liquid medium (yield extract 0.25%, peptone 0.5%, glucose 0.1%, pH7.0) was added to a 300 ml Erlenmeyer flask, and after heat sterilization, Bacillus subtilis 4-5 -1-1 strain No. 30 and Bacillus sp. 4-5-21 0306 0.1 ml of pre-culture were inoculated respectively and incubated at 28 ° C and 100 rpm in a reciprocating shaker for 3 days. Repeat the procedure of rinsing the culture solution with tap water three times to remove the cell suspension of Bacillus subtilis 4-5-1-1 strain No. 30 and Bacillus sp. 4-5-21 0306 strain. It prepared.

Example 3

Preparation of Plant Disease Control Hydration Composition

Each Bacillus cell suspension prepared in Example 2 was dried. The live cell count of the dried product was 6x10 <^10> cfu / g, 5.5 * 10 <^10> cfu / g, respectively. 10 parts by weight of each cell dried product, 2 parts by weight of lauryl sulfate sodium salt, 9 parts by weight of naphthalenesulfonic acid (sodium) formaldehyde condensate, 79 parts by weight of calcium sulfate and the hydrate thereof were uniformly mixed to obtain hydrated A and B. . Hydrating agents A and B are compositions comprising, as active ingredients, Bacillus subtilis 4-5-1-1 strain No. 30 and Bacillus sp. 4-5-21 0306 strain, respectively.

Example 4

Test on the control effect of gray mold disease of plant disease control composition (indoor test)

Each of the wetting agents A and B obtained in Example 3 was diluted 1000 times with tap water to prepare a treatment liquid. Further, as a comparative example, a commercially available boat killer hydrant (manufactured by Idemitsu Kosan Co., Ltd.) and an impression hydrant (manufactured by SDS Biotech Co., Ltd.) were diluted 1000 times and 500 times with tap water, respectively, to prepare a treatment liquid. Each treatment solution was sprayed on a kidney bean. Then, the firearms were placed on the kidney beans and incubated at 15 ° C., the low temperature region of gray mold disease, and after 24 hours, a conidia of spores of Botrytis cinerea, a pathogen of gray mold disease, 5.0 × 10 ⁵ cfu / mL) was sprayed on the fire of kidney beans. After sparging treatment, the cells were incubated at 15 ° C. for 5 days. In addition, in contrast to the untreated treatment, only the conidia of spore suspension of Botrytis cinerea (5.0 × 10 ⁵ cfu / ml) were sprayed. Irradiation measured the lesion diameter formed in the kidney bean leaf.

[Equation 1]

Control value = (1-(leak diameter of treatment tool / leave diameter of non-treatment tool)) * 100

As can be seen from Table 1, two Bacillus sp. 2 strains were confirmed to have a control effect against kidney bean gray mold disease because no lesions were identified with respect to kidney beans. On the other hand, the boat killer hydration and the impression hydration showed low control values of 19.5 and 19.8, respectively, for the kidney bean gray mold disease.

Disclosure Microorganisms Kidney bean leaf lesion measurement investigation Lesion diameter (mm) Control Bacillus subtilis 4-5-1-1 strain No.30 FERM BP-10244 0.0 100.0 Bacillus sp. 4-5-21 0306 Note FERM BP-10245 0.0 100.0 Boat Killer Hydration 30.1 19.5 Impression Hydration 30.0 19.8 No treatment 37.4

Example 5

Test of the control effect of kidney beans gray mold disease of potency control plant composition (pot test)

Each of the wetting agents A and B obtained in Example 3 was diluted 1000 times with tap water to prepare a treatment liquid. As a comparative example, the boat killer hydrant (Idemitsu Kosan Co., Ltd.) was diluted 1000 times with tap water to prepare a treatment liquid. Each treatment was sprayed into pots of kidney beans. Spraying twice every three days and grown in an incubator at 14 ° C. The day after the two sprays, conidia spore suspension (5.0 × 10 ⁵ cfu / ml) of Botrytis cinerea, a pathogen of gray mold disease, was sprayed into pots of kidney beans. Thereafter, the resultant was further grown in an incubator at 14 ° C, and after 1 week, the number of healthy and endemic fruit was measured.

As can be seen from Table 2, each Bacillus sp. 2 strain has a high control value of 86.4 and 82.5, respectively, against gray mildew disease of kidney beans, and thus, against gray mildew disease of kidney beans even in low temperature conditions. It was confirmed to have a high control effect. In contrast, the boat killer hydration had insufficient control at 14.4.

Disclosure Microorganisms Gray mold disease of kidney beans Incidence Rate Control Bacillus subtilis 4-5-1-1 strain No.30 FERM BP-10244 11.1 86.4 Bacillus sp. 4-5-21 0306 Note FERM BP-10245 14.3 82.5 Boat Killer Hydration 70.0 14.4 No treatment 81.8

Example 6

Effect of plant fungus control hydration composition of citrus fruit on gray mold disease control effect (packaging test)

Wetting agents A and B obtained in Example 3 were diluted 1000 times with tap water, and the process liquid was prepared. In addition, as a comparative example, the boat killer hydrating agent (Idemitsu Kosan Co., Ltd.) and the impression hydrating agent (SDS Biotech Co., Ltd.) were diluted 1000 times and 500 times with tap water, and the process liquid was prepared. Each treatment solution was planted in a plastic house and sprayed on the branches. The spraying treatment was performed twice at intervals of about 7 days, and after 7 days of the final spraying, the number of onset of gray mold disease and the number of whole fruits were investigated. In addition, citrus fruit infected with Botrytis cinerea as an infectious agent was dotted in the test house. In addition, the temperature during the test period was recorded.

As shown in FIG. 1, when looking at the temperature change at this time, minimum temperature is less than 15 degreeC, and even if average temperature is seen, more than half of the test period is less than 15 degreeC. In addition, days less than 10 degreeC are seen several days. Even at the highest temperature, the temperature was nearly 25 ° C at the beginning of the test, but the temperature was lower than 16 ° C-17 ° C in the middle and 15 ° C in the latter half. Of these hypothermic conditions, as can be seen from Table 3, Bacillus subtilis 4-5-1-1 strain No.30 and Bacillus sp. 4-5-21 0306 strain, eggplant gray The high control value was 68.1 and 80.4 for fungal diseases, respectively, and it was confirmed that they had a high control effect against eggplant gray mold disease even at low temperature conditions. In contrast, the control values of the boat killer hydration and the impression hydration were 53.1 and 59.5, respectively, which were not necessarily sufficient.

Disclosure Microorganisms Eggplant gray mold disease Incidence Rate Control Bacillus subtilis 4-5-1-1 strain No.30 FERM BP-10244 14.0 68.1 Bacillus sp. 4-5-21 0306 Note FERM BP-10245 8.6 80.4 Boat Killer Hydration 20.6 53.1 Impression Hydration 17.8 59.5 No treatment 43.9

Example 7

Test on the control effect of gray mold disease of plant disease control composition (indoor test)

Bacillus subtilis cultured and prepared in the same manner as in Example 2 4-5-1-1 strain No.30 FERM BP-10244 10% freeze-dried product (1.0 × 10 ¹⁰ cfu / g) and show suspension Waclay 40%, potassium chloride 40%, and nucalgen PS-P 10% were mixed uniformly, and then pulverized to obtain a wetting agent C. It was diluted 1000 times, 2000 times, and 4000 times with tap water, and the process liquid was prepared. In addition, as a comparative example, the commercially available boat killer hydrant (manufactured by Idemitsu Kosan Co., Ltd.) and the impression hydrant (manufactured by SDS Biotech Co., Ltd.) were diluted with tap water 1000 times, 2000 times, 4000 times, 500 times, 1000 times, and 2000 times, respectively. The treatment liquid was prepared. Each treatment was sprayed on a firearm of kidney beans. Subsequently, the firearm was placed on the kidney bean leaves and incubated at 15 ° C., a low temperature region of gray mold disease occurrence, and after 24 hours, a conidia of spores of Botrytis cinerea, a pathogen of gray mold disease, (5.0 10 ⁵ cfu / ml) was sprayed on the fire of kidney beans. After sparging treatment, the cells were incubated at 15 ° C. for 5 days. In addition, as an untreated treatment, only the conidia of spore suspension of Botrytis cinerea (5.0 × 10 ⁵ cfu / ml) were sprayed. Irradiation measured the lesion diameter formed in the kidney bean leaf.

As can be seen from Table 4, Bacillus subtilis 4-5-1-1 strains showed higher gray fungal control activity even when the cell concentration in the spray solution was lower than that of the boat killer or impression hydrate. It became.

Disclosure Drug Dilution factor Kidney Bean Leaf Lesion Diameter (mm) Control Bacillus subtilis 4-5-1-1 strain No.30 FERM BP-10244 1.0 × 10 ¹⁰ cfu / g × 1000 1.6 95.5 × 2000 10.6 70.1 × 4000 4.4 87.6 Boat Killer Hydration 1.0 × 10 ¹¹ cfu / g × 1000 12.0 66.1 × 2000 19.2 45.8 × 4000 29.8 15.8 Impression Hydration 5.0 × 10 ⁹ cfu / g × 500 17.8 49.7 × 1000 15.0 57.6 × 2000 23.6 33.3 No treatment - 35.4

Example 8

Citrus Ulcer Bacteria Control Effect Test of Plant Disease Control Composition (Indoor Test)

Wetting agent C used in Example 7 was diluted 1000 times with tap water, and the cut lemon leaf which wounded was immersed. After 6 hours of standing in a humidified state, a citrus ulcer bacteria suspension (A ₆₀₀ = 0.5) was added dropwise to the wound site. Incubate at 25 ° C. for 10 days. In addition, only the citrus ulcer bacteria suspension was treated dropwise with no treatment as a control. The investigation determined the incidence according to the following criteria.

1 week after inoculation

0: ulcer not detected

1: slightly verified

2: confirmed

3: remarkably confirmed

By dividing into four stages, the control value was obtained.

As can be seen from Table 5, it became clear that Bacillus subtilis 4-5-1-1 strain No. 30 has a high control activity against citrus ulcer disease.

Disclosure Drug Onset Control Bacillus subtilis 4-5-1-1 strain No.30 FERM BP-10244 2.0 91.0 No treatment 19.0

Example 9

Soft disease control effect test of plant disease control composition (indoor test)

Wetting agent C used in Example 7 was diluted 1000 times with tap water, and the lettuce slice was immersed. After 6 hours of standing in a humidified state, soft germ suspension (A ₆₀₀ = 0.5) was added dropwise to the cut portion of the section. Incubate at 25 ° C. for 7 days. In addition, only the soft germ suspension was added dropwise with no treatment as a control. The investigation calculated the incidence value by calculating the incidence according to the following criteria.

7 days after treatment

0: no lesion

1: lesion is near the inoculation site

2: lesion extends to the entire middle area but rests on the inside of the shell

3: lesions reach the outer sheath, but spread from side to side are less than 1 cm from the middle

4: lesion extends to outer shell and spreads more than 1 cm from middle

Divided into 5 steps, the control agent was obtained.

As can be seen from Table 6, it became clear that Bacillus subtilis 4-5-1-1 strain No. 30 was found to have a control effect against soft diseases of vegetables.

Disclosure Drug Onset Control Bacillus subtilis 4-5-1-1 strain No.30 FERM BP-10244 14.0 42.0 No treatment 24.0

The plant disease control composition of the present invention is suitable for various diseases such as plant diseases such as various horticultural crops, in particular, gray mold disease, fungal nucleus disease, powdery mildew, anthrax disease, rotary disease, blue mold disease, yeast mold disease, soft disease, ulcer disease, etc. On the contrary, even when a commercially available Bacillus preparation contains microorganisms having a stable and excellent control ability even under low temperature conditions, it is possible to maintain disease control ability over a long period of time, and to control other kinds of diseases at once. Can be. According to the method for controlling the plant diseases of the present invention using such a plant disease control composition, plant diseases, particularly gray It can effectively control fungal disease, fungal nucleus disease, powdery mildew, anthrax disease, rotary disease, blue mold disease, yeast mold disease, soft disease and ulcer disease.

1 is a view showing a change in temperature during the test period (Example 6).

Claims (13)

A plant disease control composition comprising a bacterium belonging to the genus Bacillus sp. Having high disease control ability under low temperature conditions in which a commercially available Bacillus preparation does not exhibit sufficient effect. The method of claim 1, Low temperature conditions are 10-15 degreeC, The plant disease control composition characterized by the above-mentioned. The method according to claim 1 or 2, Plant disease control composition, characterized in that the plant disease is at least one of gray mold disease, fungal disease, powdery mildew, anthrax disease, leap disease, blue mold disease, yeast fungus disease, soft disease, ulcer disease. The method according to any one of claims 1 to 3, Microorganism belonging to the genus Bacillus sp. Bacillus subtilis (Bacillus subtilis) 4-5-1-1 strain No. 30, characterized in that the plant disease control composition. The method according to any one of claims 1 to 3, A plant disease control composition comprising a microorganism belonging to the genus Bacillus sp., Bacillus sp. 4-5-21 0306. The method according to any one of claims 1 to 3, The microorganism belonging to the genus Bacillus sp. Is a strain mainly derived from Bacillus subtilis 4-5-1-1 strain No. 30, and has changed only a few of its characteristics. Plant pest control composition. The method according to any one of claims 1 to 3, The microorganism belonging to the genus Bacillus sp. Is a strain mainly derived from Bacillus sp. 4-5-21 0306 strain, which has changed only a few of its characteristics. . A plant disease control method using the plant disease control composition of any one of Claims 1-7, The plant and / or the soil control method characterized by the above-mentioned. Bacillus subtilis 4-5-1-1 strain No.30. Bacillus sp. 4-5-21 0306 week. Bacillus subtilis A strain mainly derived from No.30, 4-5-1-1, and which has changed only a few of its characteristics. Bacillus sp. 4-5-21 A strain mainly derived from 0306 weeks and having changed only a few of its characteristics. Next, the selection method of the microorganism belonging to the genus Bacillus sp. Which has high activity at the low temperature of Claim 1 which consists of each successive process of a-d. a. Process of collecting bacteria from strain isolate b. Process for selecting strains having a control effect against gray mold (Botrytis cinerea) at room temperature (18 ~ 25 ℃) c. Process for selecting strains that can grow at low temperature (8-12 ℃) d. Reselecting a strain having a control effect against gray mold (Botrytis cinerea) at low temperature (13-15 ℃).

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