JP2005097164A - Blight-controlling agent composition and microorganism - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a blight-controlling agent composition which constantly has an excellent controlling ability against the blight of various plants such as agricultural and horticultural crops, especially the blight of wide target plants, such as B. cinerea, sclerotal blight, mildew, anthracnose, ring spot, P. italicum, and blight caused by Penicillium digitatum. <P>SOLUTION: The blight-controlling agent composition is characterized by containing at least microorganisms which can be separated from tea leaves and belong to the Bacillus sp. and have excellent controlling abilities against B. cinerea, sclerotal blight, mildew, anthracnose, ring spot, P. italicum, and blight caused by Penicillium digitatum, as an active ingredient. The blight-controlling agent composition is used for treating plants and/or soils. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a novel microorganism belonging to the genus Bacillus having an ability to control plant diseases such as gray mold disease of various agricultural and horticultural crops, a plant disease control agent composition containing the novel microorganism, and the plant disease control The present invention relates to a method for controlling plant diseases using an agent composition.

  Conventionally, benzimidazole fungicides and thiophanate fungicides (hereinafter referred to as benzimidazole / thiophanate fungicides) have been used for various types of crops that are parasitic on agricultural and horticultural crops. It has an excellent control effect against pathogenic bacteria, and has been widely used as an agricultural and horticultural fungicide since about 1970, and has greatly contributed to increased crop production.

  On the other hand, biological pesticides are known as means that should be used in place of or in combination with control with a synthetic fungicide and that have very little environmental pollution, harmonize with the ecosystem, and have an excellent control effect. Microorganisms belonging to the genus Trichoderma, Gliocladium, and Bacillus are examples of microorganisms that have been used for disease control of such agricultural and horticultural crops, particularly gray mold disease. Many horticultural fungicide compositions have been researched and developed.

For example, regarding bacteria belonging to the genus Bacillus, JP-A-63-273470 discloses Bacillus subtilis JB3 strain (NIB12375), Bacillus subtilis JB3.6 strain (NCIBB12376), Bacillus subtilis R1 strain. Antibacterial substances obtained from fungi (NCIB12616) or mutants thereof suppress plant diseases, animal and human microbial infections, and further suppress general microbial contamination. Attempts have been made to control diseases of various agricultural and horticultural crops (see, for example, Patent Document 1). In addition, an antibacterial substance obtained from the aforementioned Bacillus subtilis JB3 strain (NCIB12375), Bacillus subtilis JB3.6 strain (NCIBB12376), Bacillus subtilis R1 strain (NCIB12616), or a mutant strain thereof is isolated, Attempts have been made to use this for disease control of various agricultural and horticultural crops (see, for example, Patent Document 2). In addition, Bacillus amyloliquefaciens is known as a microorganism for controlling mulberry anthrax and white crest feather disease (see, for example, Patent Document 3, Non-Patent Document 1, and Non-Patent Document 2).
JP-A-63-273470 JP-A-2-22299 Japanese Patent Laid-Open No. 11-246324 Plant Protection No. 56 No. 8 (2002) Phytopathology, Vol.91, No.2, 2001

  However, none of the methods for controlling diseases of agricultural and horticultural crops using bacteria belonging to the genus Bacillus is necessarily sufficient in terms of sustainability, fixability, stability and the like. In addition, all conventional methods for controlling diseases of agricultural and horticultural crops using bacteria belonging to the genus Bacillus are limited in target diseases, and in various situations where various diseases occur at the same time, it is not always possible to sufficiently control them. It was.

  The present invention has been made in view of such circumstances, and the object of the present invention is to control the plant diseases such as various agricultural and horticultural crops, in particular, the target plant diseases such as gray mold disease, with excellent control. An object of the present invention is to provide a microorganism belonging to the genus Bacillus having an ability, a plant disease control composition containing the microorganism, and a plant disease control method for treating plants and / or soil using the microorganism.

  In order to solve the above problems, the present inventors first isolated candidate microorganisms having a plant disease control ability from tea leaves collected from each field of Nippon Soda Co., Ltd. Sugawara Field Research Center. Wash the leaves of tea leaves collected from each field in a small amount of sterile water, smear the washed water on a standard agar medium (manufactured by Nissui Pharmaceutical Co., Ltd.), collect colonies of the growing microorganisms, and Separated and acquired so as to form a single colony. Among these microorganisms, three strains of microorganisms having a control effect against gray mold (B. cinerea) were found. These three strains were predicted as bacteria belonging to the genus Bacillus from the morphology of the colonies, and their morphology and mycological properties were as follows.

  Gram-positive bacilli were positive for catalase reaction, aerobically produced acid, and Bacillus-type spore formation was observed. Moreover, as a result of phylogenetic analysis and homology search based on the approximately 700 bp base sequence of 16S rDNA, strain 4-5-1 No. 30 has a homology agreement rate of 93.0% with the genus Bacillus, No. 32 is a homology agreement rate of 99.0% with Bacillus subtilis, 4-5-6 strain No. 32. 35 had a homology agreement rate of 99.0% with Bacillus subtilis. Furthermore, API 50 CH of the API manual kit (bio Merieux Industry) was used, and in all cases, the homology with Bacillus subtilis was as low as 34.4%. Furthermore, when the utilization of the carbon compound was investigated, the 4-5-1 strain No. 30 is Glycerol, L-Arabinose, Ribose, D-Xylose, D-Glucose, D-Fructose, D-Mannose, Inositol, Mannitol, Sorbitol, α-Methyl-D-glucoside, Amygdaline, Arbutin, Esculin, Salicin, Cellobiose , Maltose, Lactose, Melibiose, Sucrose, Trehalose 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, D-Arabitol, Gluconate, 2-ceto-gluconate, 5-ceto-gluconate It was not available. 4-5-3 strain no. 32 and 4-5-6 strain no. 35 is Glycerol, L-Arabinose, Ribose, D-Xylose, D-Glucose, D-Fructose, D-Mannose, Inositol, Mannitol, α-Methyl-D-glucoside, Amygdaline, Arbutin, Esculin, Salicin, Cellobiose, Maltose , Lactose, Melibiose, Sucrose, Trehalose are available, Erythritol, D-Arabinose, L-Xylose, Adonitol, β-Methyl-D-xyloside, Galactose, L-Sorbose, Rhamnose, Dulcitol, Sorbitol, α-Methyl-D -mannoside, N-Acethyl-glucosamine, Inuline, Melezitose, Xylitol, D-Turanose, D-Lyxose, D-Tagatose, D-Fucose, D-Arabitol, Gluconate, 2-ceto-gluconate, 5-ceto-gluconate It was not available.

  Based on the above results, these three strains were close to Bacillus subtilis, but the species could not be identified and Bacillus sp. Was judged. These Bacillus sp. 4-5-1 strain No. 30, Bacillus sp. 4-5-3 strain no. 32, Bacillus sp. 4-5-6 strain no. Each strain named 35 is deposited at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center under the deposit number FERM P-19458, the deposit number FERM P-19259, and the deposit number FERM P-19460.

  The present invention is a microorganism belonging to the genus Bacillus (Bacillus sp.) That can be isolated from tea leaves and has a controlling effect on plant diseases, Bacillus sp. 30 (FERM P-19458), Bacillus sp. 4-5-3 strain no. 32 (FERM P-19459) and Bacillus sp. 4-5-6 strain No. 3 35 (FERM P-19460) containing at least one selected from plant disease control composition (Claim 1) and plant diseases are gray mold, mycorrhizal disease, powdery mildew, The plant disease control agent composition according to claim 1 (claim 2) or the plant according to claim 1 or 2, which is at least one of anthracnose disease, ring spot disease, blue mold disease, and green mold disease. A plant disease control method characterized by treating a plant and / or soil with a disease control agent composition (Claim 3), Bacillus sp. 30 (FERM P-19458) (Claim 4) and Bacillus sp. 4-5-3 strain no. 32 (FERM P-19459) (Claim 5) and Bacillus sp. 4-5-6 strain No. 35 (FERM P-19460) (Claim 6).

  The plant disease control agent composition of the present invention contains a microorganism having constant and excellent control ability against various diseases such as gray mold disease, especially for plant diseases such as various agricultural and horticultural crops. It is possible to maintain the disease control ability over a long period of time and to control other types of diseases at once. According to the plant disease control method of the present invention using such a plant disease control agent composition, the plant disease control method is applied to plants such as various agricultural and horticultural crops or to the soil by a method such as spraying the plant disease control composition. In particular, it can be effectively controlled against gray mold.

  As a plant disease control agent composition of the present invention, a microorganism belonging to the genus Bacillus (Bacillus sp.) Which can be isolated from tea leaves and has a control effect against gray mold (B. cinerea), Bacillus sp. sp.) 4-5-1 strain No. 30 (FERM P-19458), Bacillus sp. 4-5-3 strain no. 32 (FERM P-19459) and Bacillus sp. 4-5-6 strain No. 3 Although it will not restrict | limit especially if it is a plant disease control agent composition characterized by containing at least 1 sort (s) chosen from 35 (FERM P-19460), The microorganisms which belong to the said Bacillus genus (Bacillus sp.) However, those having a controlling effect against C. lagenarium, P. longiseta, citrus green mold (P. digitatum), citrus green mold (P. italicum) and the like are preferable. .

  The plant disease control agent composition of the present invention can be particularly advantageously used against gray mold, mycorrhizal disease, powdery mildew, anthrax, ring spot disease, blue mold disease, green mold disease and the like.

  In the production of the plant disease control agent composition of the present invention, it is possible to use cells obtained by growing the microorganism belonging to the genus Bacillus of the present invention by a known means such as solid culture or liquid culture. it can. As a method for growing such microorganisms, any method may be used as long as the cells grow, regardless of the type of medium, culture conditions, etc. For example, in the case of solid culture, a standard agar medium or ordinary agar is used. In the case of liquid culture, static culture at 25 to 35 ° C. in a medium, potato dextrose agar medium or the like, and shaking culture at 25 to 35 ° C. in various liquid media obtained by removing agar from the agar medium can be mentioned. . In addition to the cells themselves, the cells may be used in any form such as suspensions or culture solutions or concentrates, pastes, dried products, diluted products, etc. Can do.

The concentration of the bacterium belonging to the genus Bacillus (Bacillus sp.) In the disease control agent composition of the present invention is not particularly limited, but when diluted 1000 to 2000 times, it is converted into the cell concentration, A range of 1 × 10 ¹¹ to 1 × 10 ² cfu / ml, preferably 1 × 10 ⁹ to 1 × 10 ⁴ cfu / ml can be suitably exemplified. The disease control agent composition of the present invention can be mixed with commonly used carriers, surfactants, dispersants, adjuvants, and the like, and the form can be taken by ordinary agricultural chemicals, for example, powders. , Wettable powders, emulsions, flowables, granules, capsules and the like can be employed.

  Examples of the carrier include solid carriers such as diatomaceous earth, clay, talc, bentonite, white carbon, kaolin, vermiculite, slaked lime, quartz sand, ammonium sulfate, and urea. Surfactants and dispersants include, for example, alkylphenyl ethers added with polyoxyethylene, alkyl ethers added with polyoxyethylene, higher fatty acid esters added with polyoxyethylene, sorbitan higher fatty acid esters added with polyoxyethylene , Nonionic surfactants such as tristyryl phenyl ether added with polyoxyethylene, sulfates of alkylphenyl ether added with polyoxyethylene, metal salts of alkylbenzene sulfonate, sulfates of higher alcohols, alkyl naphthalene sulfone Ionic surfactants and dispersants such as sodium acid, sodium lignin sulfonate, formaldehyde condensates of sodium alkylnaphthalene sulfonate, and copolymers of isobutylene-maleic anhydride It can be mentioned. Examples of the auxiliary agent include carboxymethyl cellulose, polyethylene glycol, gum arabic, starch, and lactose.

  In order to produce the disease control composition of the present invention as an emulsion, a suspension in which collected and dried Bacillus sp. Cells are mixed in an organic solvent containing a surfactant is prepared. This can be done. Any surfactant can be used as long as it does not inhibit the growth of bacterial cells. Specifically, polyoxyethylene sorbitan monolaurate, polyoxyethylene glycol monolaurate And polyethylene glycol monooleate. These may be used alone or in combination of two or more. Examples of organic solvents include vegetable oils such as soybean oil, rapeseed oil, castor oil, cottonseed oil, palm oil, safflower oil, spindle oil, heavy white oil, light white oil, mineral spirit, mineral turpentine, naphthenic oil, paraffin. Examples thereof include mineral oils such as oil, machine oil for agricultural chemicals, silicone oil, and the like, and these can be used alone or in combination of two or more.

The method for controlling plant diseases of the present invention is not particularly limited as long as it is a method for controlling plant diseases using the plant disease control composition of the present invention, and the plant disease control composition of the present invention. In the same manner as ordinary chemical pesticides, there can be mentioned a method of spraying on plants and soil such as various agricultural and horticultural crops. In the spraying treatment, the plant disease control agent composition of the present invention can be used by diluting with an appropriate amount of water or the like, and the spraying amount is converted to the concentration of cells belonging to the genus Bacillus sp. In general, it can be in the range of 1 × 10 ¹¹ to 1 × 10 ² cfu / ml, preferably 1 × 10 ⁹ to 1 × 10 ⁴ cfu / ml.

  Hereinafter, the plant disease control agent composition of the present invention and the plant disease control method using the same will be specifically described with reference to examples, but the present invention is not limited to the following examples. As long as it does not deviate from the above, the kind of bacteria of the genus Bacillus (Bacillus sp.), The composition ratio of the preparation, the dosage form and the like can be freely changed. Moreover, in the following Examples, Bacillus sp. 4-5-1 strain No. as Bacillus sp. 30 (FERM P-19458), Bacillus sp. 4-5-3 strain no. 32 (FERM P-19459), Bacillus sp. 4-5-6 strain no. 35 (FERM P-19460) was used.

(Preparation of Bacillus sp. Cell suspension)
A 300 ml Erlenmeyer flask was charged with 150 ml of a standard liquid medium (yeast extract 0.25%, peptone 0.5%, glucose 0.1%, pH 7.0), sterilized by heating, and then Bacillus sp. 4-5 -1 stock No. 30, Bacillus sp. 4-5-3 strain no. 32, Bacillus sp. 4-5-6 strain no. Each of the 35 precultures (0.1 ml) was inoculated and cultured in a reciprocating shaker at 28 ° C. and 100 rpm for 2 days. The operation of washing the culture solution with tap water was repeated three times, and finally Bacillus sp. 4-5-1 strain No. 1 so as to be 1 × 10 ¹¹ cfu / ml. 30, Bacillus sp. 4-5-3 strain no. 32, Bacillus sp. 4-5-6 strain no. 35 bacterial cell suspensions were prepared.

(Manufacture of plant disease control wettable powder composition)
Each Bacillus sp. Cell suspension prepared in Example 1 (1 × 10 ¹¹ cfu / ml) 2 parts by weight cell pellet, Showa clay 45%, potassium chloride 45%, Newkalgen PS-P 7% 1 part by weight of a white sample (excluding only the active ingredient from the preparation) was uniformly mixed, dried, and pulverized to obtain wettable powders A, B, and C. The wettable powders A, B, and C were obtained from Bacillus sp. 30, Bacillus sp. 4-5-3 strain no. 32, Bacillus sp. 4-5-6 strain no. It is a composition containing 35 as an active ingredient.

(Control effect test of plant disease control wettable powder composition; laboratory test)
Each wettable powder A, B, C obtained in Example 2 was diluted 1000 times with tap water to prepare a treatment solution. The treatment solution was sprayed on the flower vase. Thereafter, the mixture was incubated at 20 ° C., and after 24 hours, a conidial spore suspension (5.0 × 10 ⁵ cfu / ml) of Botrytis cinerea, a pathogen of gray mold disease, was sprayed on the kidneys of kidney beans. . After the spraying treatment, it was incubated at 20 ° C. for 3 days. In addition, as a control, only a conidial spore suspension (5.0 × 10 ⁵ cfu / ml) of Botrysis cinerea was sprayed. Survey: 0: No growth of B. cinerea hyphae (no disease), 1: A little growth of hyphae is observed, 2: Mycelium growth is less than 50%, 3: Hyphae throughout the vase The four stages of growth were observed.

Disease severity = Σ (index × number of corresponding vases) / (number of surveyed vases × 3) × 100
Control value = (1- (Disease level of treated area / Disease level of untreated area)) × 100
Moreover, the bean leaf was laid under the bean vase and the lesion diameter was measured.

  As is clear from Table 1, each of the three strains of Bacillus sp. (Bacillus sp.) Has no disease and lesions on the organs and leaves of green beans, and has a control effect on green bean mold. Was recognized.

(Control effect test of plant disease control hydrating composition; field test)
Each wettable powder A and B obtained in Example 2 was diluted 1000 times with tap water to prepare a treatment solution. The treatment liquid was sprayed on strawberries planted in a vinyl house. Spraying was performed three times at intervals of about 10 days, and the number of gray mold diseases and the number of healthy fruits were investigated 10 days after the final spraying, and the control results were calculated. Citrus fruits infected with Botrytis cinerea as an infection source were scattered in the test house.

  As is apparent from Table 2, the Bacillus sp. 4-5-1 strain No. 30 and the Bacillus sp. 4-5-3 strain no. As for 32, the high control value with 94.4 and 72.3 was obtained with respect to strawberry gray mold disease, respectively, and it was recognized that it has a high control effect with respect to strawberry gray mold disease.

(Control effect test of plant disease control hydrating composition; field test)
The wettable powder C obtained in Example 2 was diluted 1000 times with tap water to prepare a treatment liquid. The treatment liquid was sprayed on strawberries planted in a vinyl house. Spraying was performed three times at intervals of about 10 days, and the number of gray mold diseases and the number of healthy fruits were investigated 10 days after the final spraying, and the control results were calculated. Citrus fruits infected with Botrytis cinerea as an infection source were scattered in the test house.

  As is apparent from Table 3, Bacillus sp. 4-5-6 strain no. 35 was found to have a high control value of 70.2 against strawberry gray mold disease, and was found to have a high control effect against strawberry gray mold disease.

(Control effect test of plant disease control hydrating composition; field test)
The wettable powder B obtained in Example 2 was diluted 1000 times with tap water to prepare a treatment liquid. The treated solution was sprayed to citrus (ordinary Wenzhou) planted in the field. Fruits were collected 3 days after spraying, and the asphalt slope was rolled 10 m after collection, and the fruit surface was damaged (Results of practical application test of new pesticides in 2002 (Evergreen fruit trees)-39th (2002)- According to the method described in Japanese Plant Protection Association P.59 and P.61). Thereafter, a spore suspension of Penicillium digitatum was spray-inoculated at 5.0 × 10 ⁴ cfu / ml. After that, it was put in a box of foamed polystyrene, shielded from light with cold water, and after 8 days of drug treatment, the number of fruits and the number of healthy fruits was investigated and the control value was calculated.

  As is clear from Table 4, Bacillus sp. 4-5-3 strain No. No. 32 was found to have an extremely high control value of 93.5 against citrus green mold disease and was found to have a control effect against citrus green mold disease.

Claims (6)

A microorganism belonging to the genus Bacillus sp. That can be isolated from tea leaves and has a controlling effect on plant diseases, Bacillus sp. 4-5-1 strain No. 30 (FERM P-19458), Bacillus sp. 4-5-3 strain no. 32 (FERM P-19459) and Bacillus sp. 4-5-6 strain No. 3 A plant disease control composition comprising at least one selected from 35 (FERM P-19460). The plant disease control agent composition according to claim 1, wherein the plant disease is at least one of gray mold disease, mycorrhizal disease, powdery mildew disease, anthrax disease, ring spot disease, blue mold disease, and green mold disease. Stuff. A plant disease control method comprising treating a plant and / or soil with the plant disease control agent composition according to claim 1 or 2. Bacillus sp. 4-5-1 strain No. 30 (FERM P-19458). Bacillus sp. 4-5-3 strain no. 32 (FERM P-19459). Bacillus sp. 4-5-6 strain No. 35 (FERM P-19460).