JP2019189553A - Production method of microbial pesticide - Google Patents

Abstract

To provide, in production of a microbial pesticide in which Pseudomonas microbe is made a microbe having control ability, a culture method of microorganisms for a microbial pesticide having excellent characteristics, as cultured microorganisms for producing a microbial pesticide, by securing an effective viable cell count for securing the efficacy as the pesticide during microbial pesticide application, and by preventing a decrease of the viable count and degradation of the storage stability in the treatment during formulation of micro organisms of microbial pesticide, the circulation, and storage.SOLUTION: By adopting a culture method that adopts two culture temperature conditions in which a cultured microorganism for producing a microbial pesticide is cultured at a normal temperature as a first culture, then, is cultured under a low temperature of 5 to 15°C as a second culture, cultured microorganisms having excellent viable cell survivability, and storage stability to the treatment during formulation of micro organisms of microbial pesticide, the circulation, and storage are provided.SELECTED DRAWING: None

Description

  The present invention relates to a microbial pesticide using a Pseudomonas bacterium as a microorganism having a pesticide control ability. Specifically, in a microbial pesticide using a Pseudomonas bacterium as a microorganism having a pesticide control ability, Culturing by the first culture cultivated at a culture temperature of 2 and then the second culture cultivated at a low temperature of 5 to 15 ° C., and drying using the cultured microorganism, Further, the present invention relates to a method for producing a microbial pesticide which ensures the number of viable microorganisms and the storage stability for preservation. In the method for producing a microbial pesticide of the present invention, Pseudomonas bacteria used as a microorganism having a pesticide control ability include Pseudomonas azotoformans W-14-1 strain (Pseudomonas azotoformans NITE BP-02371). be able to.

  In pesticides for controlling plant pathogens, in order to overcome the problems of resistant bacteria and phytotoxicity, instead of conventional synthetic fungicides, or as a means to use in combination to reduce the harmful effects of synthetic fungicides, Conventionally, interest in microbial pesticides has increased, and various studies have been conducted on the use of microbial pesticides. Microbial pesticides have advantages such as extremely low environmental pollution, harmony with the ecosystem, and excellent control effect compared to conventional synthetic fungicides. However, since microbial pesticides use living organisms as ingredients with pesticide control, ensuring the activity of bacteria necessary to show the effect as a pesticide when applying microbial pesticides and ensuring stability against storage are problems. Therefore, conventionally, the development of new active strains, the formulation of microbial pesticides, the distribution of microbial pesticides, and the stability during storage have been studied.

  In relation to microbial pesticides, there are long-standing research reports on the storage stability of microorganisms. For example, low-molecular substances such as amino acids, organic acids, and sugars, proteins, polysaccharides, and synthetics have been used as storage protection agents. A method for improving storage by adding a high-molecular substance such as a polymer and freeze-drying has been reported (Non-patent Document 1).

  In the microbial pesticide, a representative microorganism used as a component for controlling pathogenic bacteria of the pesticide includes Pseudomonas bacteria, but the microbial pesticide is applied when the microbial pesticide is formulated using the microorganism. In order to ensure the effectiveness as a pesticide at the time, in addition to searching for active effective microorganisms, the activity of the microorganism pesticides during the formulation of the microorganisms, the reduction in activity and the stability during distribution and storage Various methods are disclosed as a response to the above. For example, Patent Document 1 discloses a method for producing a microbial pesticide having microbial cells adsorbed on a zeolite base material, air-dried, and having activity and stability as viable bacteria. In microbial pesticides of rice seedlings that use Pseudomonas bacteria as microorganisms that have the ability to control pesticides, when formulating the microbial pesticides, the strain is mixed with sugars such as sucrose, fructose, glucose, sorbitol, and vacuum A method for stably immobilizing a strain while maintaining high viability by lyophilization or vacuum drying is disclosed.

  In Patent Document 3, a bag containing an adsorbent having ammonia adsorption ability such as zeolite, molecular sieve, and silica gel is added to a microbial pesticide preparation. As a method for preserving the preparation while maintaining the survival rate and activity of microorganisms necessary for exhibiting the effect as described above, Patent Document 4 discloses that a mixture of Pseudomonas bacteria is mixed with trehalose, and is frozen and then vacuum dried. A method for producing a microbial pesticide capable of providing a large amount of immobilized products having excellent pulverizable physical properties and good viability recovery rate and storage stability is disclosed in Patent Document 5, At the time of formulation, (A) sodium chloride, potassium chloride, and / or (B) trehalose and sucrose are mixed in a microbial cell suspension capable of controlling agrochemicals, and the mixture is freeze-dried. It allows the number of viable bacteria in microbial pesticide, a method for stable maintenance is disclosed a long period of time.

  Furthermore, Patent Document 6 discloses that in the preparation of a microbial pesticide, the preparation is prepared as a microbial pesticide composition comprising a microorganism having an effect of controlling plant diseases and calcium sulfate, and thus, storage stability, Regarding a method for producing a microbial pesticide composition having an antifouling effect when sprayed, Patent Document 7 describes a method for producing freeze-dried cells in a microbial pesticide, etc., an aqueous solution of trehalose and other sugars, or A method is disclosed in which a microorganism is suspended in an aqueous solution of sucrose and other sugars and freeze-dried to produce a freeze-dried cell having a high survival rate of the microorganism even after long-term storage.

  As described above, when formulating a microbial pesticide, in order to ensure its effectiveness as a pesticide at the time of microbial pesticide application, in addition to searching for active effective microorganisms, processing and distribution of the microbial pesticide at the time of formulation of the microorganism Various methods have been disclosed as countermeasures against the decrease in activity and stability during storage, but the active ingredient of microbial pesticides is microorganisms, so the effectiveness as pesticides when applying microbial pesticides In order to provide a microbial pesticide that ensures its effectiveness, it is necessary to provide a microbial pesticide that retains an effective amount of an active microbe that can exhibit its effectiveness as a pesticide in the formulation. In the production of agricultural chemicals, it is necessary to prepare cultured microorganisms having excellent characteristics that are excellent in maintaining activity and are viable and stable for storage.

  In other words, microorganisms used for microbial pesticides are usually produced by culturing, but in the production of microbial pesticides by this culture, it is necessary to make the culture conditions suitable for each microorganism, and the microorganisms were cultured according to the culture conditions. It is influenced by the activity, viability and stability of microorganisms. In particular, in the case of culturing a large amount of microorganisms for the production of microbial pesticides, the influence on the characteristics of the cultured microorganisms is large, and it is necessary to carefully design the culture conditions. For example, depending on the culture conditions, it may affect the subsequent storage stability and cause a decrease in viable bacteria. It is considered that the reduction of viable bacteria, which is a problem in microbial pesticides, is affected by the culture time, culture temperature, contamination of bacteria, growth of seed microorganisms, and the like.

  As described above, in the production of microbial pesticides, in order to ensure the effectiveness as a pesticide at the time of microbial pesticide application, in addition to securing the number of effective viable bacteria, the treatment and distribution of microorganisms of microbial pesticides, There is a need to prevent a decrease in the number of viable bacteria and storage stability during storage. For this purpose, it is important to culture and prepare cultured microorganisms with excellent characteristics of viability and storage stability. Become. Therefore, the development of a method for culturing microorganisms that satisfy the needs is an important issue for providing a microbial pesticide that ensures its effectiveness as a pesticide when applied.

JP 63-227507 A Japanese Patent Laid-Open No. 11-276166 JP 2000-264807 A JP 2005-325077 A JP 2009-196920 A JP 2011-184370 A JP 2011-223990 A

Edited by Yoshio Nei, “Preservation Method of Microorganisms” (University of Tokyo Press), 1977.

  An object of the present invention is to produce a microbial pesticide using a Pseudomonas bacterium as a microorganism having a pesticide control ability. As a method for culturing a microorganism used in the production of the microbial pesticide, the cultured microorganism is used as a pesticide at the time of applying the microbial pesticide. In addition to securing the number of viable bacteria effective to ensure the effectiveness of microbial pesticides, prevent the decrease in the number of viable bacteria and storage stability in the treatment, distribution, and storage of microorganism pesticides during the formulation of microorganisms, An object of the present invention is to provide a method for culturing microorganisms for microbial pesticides having excellent characteristics as cultured microorganisms for the production of microbial pesticides.

  In order to solve the above-mentioned problems, the present inventors, in the production of a microbial pesticide using a Pseudomonas bacterium as a microorganism having a pesticide control ability, the effectiveness of the cultured microorganism as a pesticide at the time of microbial pesticide application. Characteristics that can prevent the decrease in the number of viable bacteria and storage stability during the formulation, distribution, and storage of microorganism pesticides, as well as the number of viable bacteria effective for ensuring As a first culture, a cultured microorganism for producing a microbial pesticide is cultured at a normal culture temperature, and then as a second culture, 5-15. By adopting a culture method that employs two culture temperature conditions of culturing at a low temperature of ℃, viability of viable bacteria for treatment, distribution, and storage of microbial pesticides at the time of formulation. And maintenance It found that can be provided an excellent culture microorganisms in stability, and have completed the present invention.

  That is, the present invention relates to a method for producing a microbial pesticide using a Pseudomonas bacterium as a microorganism having a pesticide-controlling ability, culturing the Pseudomonas bacterium as a first culture at a normal culture temperature, As a culture of culturing at a low temperature of 5 to 15 ° C., using the cultured microorganisms, drying and formulating, ensuring the number of viable bacteria of active microorganisms and storage stability, It consists of a method for producing microbial pesticides. As described above, the cultured microorganism of the present invention becomes a cultured microorganism having characteristics excellent in viability and storage stability for viability and storage stability with respect to treatment, distribution, and storage during the preparation of microorganisms of microbial pesticides. By using a microorganism, a microbial pesticide having microbial activity excellent in effectiveness as a pesticide at the time of microbial pesticide application is provided.

  In the method for producing a microbial pesticide using the Pseudomonas bacterium of the present invention as a microorganism having a pesticide control ability, a temperature of 20 to 40 ° C. is used as a normal culture temperature in the first culture of the Pseudomonas bacterium. Can do. In addition, as the culture time in the first culture and the second culture, the culture time of the first culture is 1 to 50 hours, and the culture time of the second culture is 1 to 50 hours. Can be adopted.

  In the method for producing a microbial pesticide of the present invention, freeze-vacuum drying or pulse combustion drying can be used as a method for drying cultured microorganisms when dried using cultured microorganisms.

  The method for producing a microbial pesticide according to the present invention can be applied to a method for producing a microbial pesticide using a Pseudomonas bacterium as a microorganism having a pesticide-controlling ability. In the case of W-14-1 strain (Pseudomonas azotoformans NITE BP-02371), which is a kind of Pseudomonas bacteria, the culture conditions may affect the storage stability of lyophilized microorganisms. However, the method for producing a microbial pesticide of the present invention can be applied to the strain to produce a microbial pesticide that ensures the number of viable microorganisms and ensures storage stability. it can.

That is, the present invention specifically includes the following inventions.
[1] In a method for producing a microbial pesticide using a Pseudomonas bacterium as a microorganism having a pesticide-controlling ability, the Pseudomonas bacterium is cultured as a first culture at a normal culture temperature, and then as a second culture. Cultivating at a low temperature of 5 to 15 ° C., drying and formulating using the cultured microorganism, and ensuring the number of active microorganisms viable and storage stability, Production method.
[2] The method for producing a microbial pesticide according to the above [1], wherein the normal culture temperature in the first culture is 20 to 40 ° C.
[3] The microbial pesticide according to the above [1] or [2], wherein the culture time of the first culture is 1 to 50 hours and the culture time of the second culture is 1 to 50 hours Manufacturing method.
[4] The method for producing a microbial pesticide according to any one of the above [1] to [3], wherein the cultured microorganism is dried by freeze vacuum drying or pulse combustion drying.
[5] The Pseudomonas genus which is a microorganism having a pesticide-controlling ability is Pseudomonas azotoformans W-14-1 strain (Pseudomonas azotoformans NITE BP-02371). 5. The method for producing a microbial pesticide according to any one of 4 above.

  The present invention relates to the production of a microbial pesticide using a Pseudomonas bacterium as a microorganism having a pesticide-controlling ability. The microorganism used for the production of the microbial pesticide is cultured by the culture method of the present invention so that the microorganism cultured at the time of application of the microbial pesticide In addition to securing the number of viable bacteria effective to ensure the effectiveness as a pesticide, the number of viable bacteria and the storage stability are reduced for the treatment, distribution and storage of microbial pesticides when formulating microorganisms. Prepared as cultured microorganisms with excellent characteristics that prevent the formation of microorganisms, and formulated microbial pesticides using these microorganisms, they are viable and have excellent storage stability and are effective as pesticides when applied. Provided is a microbial pesticide having excellent microbial activity.

  The present invention relates to a method for producing a microbial pesticide using a Pseudomonas bacterium as a microorganism having a pesticide control ability, wherein the Pseudomonas bacterium is cultured as a first culture at a normal culture temperature, and then a second culture. As a microbial pesticide, which ensures the number of viable microorganisms and preserves the storage stability, characterized by culturing at a low temperature of 5 to 15 ° C., drying and formulating using the cultured microorganisms It consists of the manufacturing method.

(Microorganism used in the present invention)
In the method for producing a microbial pesticide according to the present invention, Pseudomonas bacteria are used as microorganisms having a pesticide control ability. Microorganisms can be classified according to whether the carbon source, which is a nutrient source, is organic carbon or carbon dioxide, and can also be classified based on whether the energy source is oxidation of a compound or light. The microorganism used in the present invention is a microorganism (chemically synthesized heterotrophic organism) in which the nutrient source is organic carbon and the energy source is classified as compound oxidation. Common algae and the like are those in which the nutrient source is only carbon dioxide and the energy source is light (photosynthesis autotrophic organism), and are not microorganisms that fall under the microorganism cultivation method of the present invention.

  As these microorganisms, Pseudomonas species are known. Further, Azotoformans sp. And the like are known as Pseudomonas species. Further, Pseudomonas azotoformans W-14-1 strain (hereinafter simply referred to as “W-14-1 strain”) can be used as the azotoformans inoculum. Pseudomonas azotoformans W-14-1 strain was established on October 12, 2016, and is based on the Patent Microorganism Deposit Center (NPMD), National Institute of Technology and Evaluation, 2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture 122 Is deposited internationally under the international deposit number NITE BP-02371.

(Viable count)
In the present invention, the viable cell count indicates the number of bacteria grown under a certain condition, and can be measured by a colony formation method using a standard agar medium. Cfu is an abbreviation for Colony Forming Unit, and indicates the number of units capable of forming a colony.

(Culture method)
In the present invention, culturing means giving a substrate and growing it, and fermentation means changing an organic substance by an enzyme produced by a microorganism, and culturing and fermentation are different. A batch culture method is used as the culture method of the present invention. The batch culture method comprises a method in which microorganisms and a medium are placed in a culture tank, the factors affecting the growth of the microorganisms such as temperature, pH, oxygen concentration, and moisture are controlled, the microorganisms are grown and discharged as a culture solution.

  In batch culture, the cell concentration of microorganisms changes over time, but the number of cells does not increase but the cell size increases, the logarithmic phase where the number of cells doubles at regular intervals, the generation time The cell concentration changes depending on the growth decay period from the start of growth until the cell division is completely stopped, the stationary phase in which the nutrients of the medium are depleted and the cell division is completely stopped, and the death period in which the number of cells starts to decrease. In the culture method of the present invention, the period until the growth decay period is distinguished for convenience from the “first culture” and the subsequent periods are classified as “second culture”.

  In the method for producing a microbial pesticide of the present invention, in the cultivation of microorganisms to be used, the temperature differs between the first culture temperature (the temperature of the first culture) and the second culture temperature (the temperature of the second culture). The second culture temperature is set lower than the first culture temperature. In the present invention, the second culture temperature is set lower than the first culture temperature, and the temperature used for the culture of Pseudomonas bacteria is usually adopted as the temperature of the first culture. Specifically, the first culture temperature is 20 to 40 ° C., preferably the first culture temperature is 25 to 35 ° C., and the second culture temperature is 5 to 15 ° C., preferably the second culture temperature. A temperature of 5-13 ° C is employed.

  In the method for producing a microbial pesticide of the present invention, the pH of the culture solution used for culturing the microorganism used is adjusted to a range of 6.0 to 8.0, preferably a range of 6.5 to 7.5, Preferably, it is adjusted to a range of 6.8 to 7.2. The optimum pH of the microorganism to be used can be applied as the pH of the culture solution.

(Substrate, medium)
A substrate is a nutrient source for microorganisms, and a culture medium contains a substrate. However, the substrate and the culture medium are not particularly distinguished, and indicate a substance containing a carbon source, a nitrogen source, an inorganic substance, or the like capable of growing microorganisms.

  In the method for producing a microbial pesticide according to the present invention, a solid medium or a liquid medium can be used as a medium for culturing microorganisms to be used. The solid medium is not particularly limited as long as it is a medium used for culturing microorganisms. Main grains such as rice and wheat, cereals such as corn, chestnut, straw, sorghum and buckwheat, oga flour, Examples include bagasse, rice husk, bran, rice bran, rice bran, corn cob, cottonseed meal, okara, agar, gelatin and the like.

  The liquid medium is not particularly limited as long as it is a medium used for culturing microorganisms. Examples of components contained in the liquid medium include monosaccharides such as glucose (glucose) and fructose (fructose) galactose, and maltose (maltose). ), Disaccharides such as sucrose (sucrose), lactose (lactose), starch, amylose, amylopectin, glycogen, cellulose, chitin, agarose, carrageenan, heparin, hyaluronic acid, pectin, xyloglucan, glucomannan, dextrin, etc. Examples include saccharides, meat extract, malt extract, yeast extract, peptone, polypeptone, dry yeast, soy flour, sodium chloride, potassium citrate and the like.

  Examples of the medium include 2% malto extract liquid medium, oatmeal liquid medium, potato dextrose liquid medium, Sabouraud liquid medium, and L-broth liquid medium.

(Culture medium)
The culture solution is a solution after culturing, and is a solution containing a cultured microorganism, a substrate, a medium, a compound produced by the microorganism, and the like.

(Culture tank)
The culture tank is not particularly limited as long as it is an apparatus used for culturing, but an Erlenmeyer flask, a jar fermenter, a bioreactor, etc. can be exemplified, and an appropriate apparatus can be used depending on the production scale. . During the culture, stirring, aeration, temperature control, pH control and the like can be performed.

(Drying method)
In the method for producing a microbial pesticide according to the present invention, dried cells can be obtained by drying a culture solution of cultured microorganisms. By drying, storability is improved and operability is improved when a preparation is made. The drying method is not particularly limited, and a commonly performed method for drying a culture solution can be used, and examples thereof include a freeze vacuum drying method and a pulse combustion type drying method. A freeze vacuum drying method can be mentioned. In the present application, the dried cells dried by freeze-drying can be called freeze-dried products or simply dried products.

(Cultured microorganisms)
The microorganisms cultured by the culturing method of the present invention can be used as microorganisms having a pesticide-controlling ability in a method for producing a microbial pesticide, thereby providing a microbial pesticide having an excellent control function. The dried cells obtained by drying the microorganisms cultured by the culture method of the present invention by freeze-drying or the like can be used for various purposes as lyophilized products containing highly active cultured microorganisms or simply as dried products. .

(Formulation)
In the method for producing a microbial pesticide of the present invention, a known method for producing a microbial pesticide can be used except that the microorganism cultured by the culture method of the present invention is used as a microorganism having a pesticide-controlling ability. In formulating the microbial pesticide, known materials and components used for the formulation can be used. That is, pesticides such as carriers, desiccants, adjuvants, surfactants or dispersants, antioxidants, colorants, lubricants, UV inhibitors, antistatic agents, preservatives, etc., used in the formulation of microbial pesticides Materials, components used for formulation, and optional components generally used in agrochemical formulation can be used.

  Carriers include inorganic salts such as calcium carbonate, potassium chloride, sodium sulfate, calcium sulfate, and ammonium sulfate; organic acids such as citric acid, malic acid, and stearic acid; and salts thereof; sugars such as glucose, lactose, and sucrose; alumina powder Solid carriers such as 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.

  Chemical desiccants such as quick lime, type III anhydrous gypsum, calcium chloride, diphosphorus pentoxide, sodium hydroxide, potassium hydroxide, anhydrous sodium sulfate, anhydrous copper sulfate, magnesium perchlorate; silica gel And physical desiccants such as aluminum oxide, molecular sieve, allophane, and zeolite.

  Examples of the auxiliary agent include carboxymethyl cellulose, polyethylene glycol, gum arabic, and starch. Examples of the surfactant or dispersant include nonionic surfactants, anionic surfactants, cationic surfactants, and amphoteric surfactants.

  In the method for producing a microbial pesticide of the present invention, trehalose, saccharose, fructose, glucose, used as an optional ingredient for formulation, used for formulation treatment of microbial pesticide, and stabilization of microbial activity in distribution and storage, Components such as saccharides such as sorbitol, inorganic salts such as sodium chloride, potassium chloride, and calcium sulfate, or adsorbents such as zeolite, molecular sieve, and silica gel can be used as appropriate.

  The microbial pesticide produced by the method for producing a microbial pesticide of the present invention or a preparation containing the microbial pesticide of the present invention can be used as a microbial pesticide for various plant disease control. Examples of the plant diseases include, for example, scab disease, perforation bacterial disease, soft rot disease, spotted bacterial disease, black spot bacterial disease, bacterial wilt disease, brown spot bacterial disease, stem rot bacterial disease, rice wilt bacterial disease, seedling establishment Bacterial diseases such as bacterial blight, white leaf blight, rot and black rot, as well as fungal diseases such as gray mold, powdery mildew, leaf mold, subtilis, black scab and scab Is mentioned.

  The microbial pesticide produced by the method for producing a microbial pesticide of the present invention or the preparation containing the microbial pesticide of the present invention can be applied alone as the microbial pesticide, but the microbial pesticide or the microorganism of the present invention It can also be applied as a pesticide in which a chemical pesticide such as a bactericide, insecticide, herbicide, growth regulator and the like is further mixed with a preparation containing the pesticide.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.

[Exemplary culture example 1]
2.5 g of yeast extract, 5.0 g of polypeptone, and 1.0 g of glucose were added to prepare 1 L of medium, which was adjusted to pH 7.0 with 1N aqueous sodium hydroxide solution. The W-14-1 strain was inoculated into this medium and cultured with shaking at 30 ° C. for 24 hours to prepare a preculture solution.

Next, 300 g of Bacterio-N KN (manufactured by Maruha Nichiro Foods Co., Ltd.), 150 g of Bacterio-N SS (manufactured by Maruha Nichiro Foods Co., Ltd.) and 75 g of glucose are added to prepare 5 L of medium, which is made of 1N sodium hydroxide aqueous solution. The pH was adjusted to 7.0. The medium was inoculated with 20 ml of the preculture solution and cultured at 30 ° C., aeration volume 1 VVM (volume per volume per minute) for 24 hours. Thereafter, the mixture was cooled to 10 ° C. over 4 hours at 5 ° C./hour, and further cultured at 10 ° C. for 20 hours. The viable cell count after the culture was 3.3 × 10 ¹⁰ cfu / ml.

Centrifuge 800 ml of the culture solution and dilute the separated pellet with 1% aqueous sodium chloride solution, and adjust the OD600 value (OD value at 600 nm, OD value is logarithm of permeability) to 250. did. To 100 ml of this diluted solution, 25 g of trehalose dihydrate was added, poured into a tray to a thickness of about 1 cm, then frozen at −60 ° C. and dried under reduced pressure (freeze drying) at a shelf temperature of 30 ° C. As a result, the viable cell count of the freeze-dried products of Example Culture Example 1 and Comparative Culture Example 1 was 3.6 × 10 ¹¹ cfu / g.

[Comparative culture example 1]
Incubation example 1 “30 ° C., aeration rate 1 VVM, 24 hours culture, then cooled to 10 ° C. over 4 hours at 5 ° C./hour, and culture at 10 ° C. for 28 hours” “30 ° C., aeration rate The culture was performed in the same manner as in Example 1 except that the culture was changed to “1 VVM, 33 hours culture”. The viable cell count after the culture was 2.8 × 10 ¹⁰ cfu / ml. The viable count of the freeze-dried product was 5.1 × 10 ¹¹ cfu / g.

[Accelerated test]
The freeze-dried products of Example 1 and Comparative Example 1 were each placed in a moist catch packaging and stored at 37 ° C. for 7 days. Thereafter, as a result of measuring the viable cell count, the viable cell counts of the freeze-dried products of Example 1 and Comparative Culture Example 1 were 2.5 × 10 ¹¹ cfu / g and 6.5 × 10 ⁸ cfu / g. It was. This value corresponds to 69% and 0.1%, respectively, compared to before the acceleration test.

(result)
As a result of the accelerated test, the culture method of W-14-1 strain was cultured at 30 ° C. for 24 hours and then cooled to 10 ° C., so that the survival rate in the subsequent accelerated test was greatly improved and storage stability was improved. It was possible to obtain a dried product excellent in

The present invention relates to the production of a microbial pesticide using a Pseudomonas bacterium as a microorganism having a pesticide-controlling ability. In addition to securing the number of viable bacteria effective to ensure the effectiveness as a pesticide, the number of viable bacteria and the storage stability are reduced for the treatment, distribution and storage of microbial pesticides when formulating microorganisms. Prepared as a cultured microorganism with excellent characteristics, which prevents the formation of microbial pesticides, and formulated as a microbial pesticide, it is effective as a pesticide at the time of pesticide application, with excellent viability and storage stability Provided is a microbial pesticide having excellent microbial activity.

Claims (5)

  In a method for producing a microbial pesticide using a Pseudomonas bacterium as a microorganism having a pesticide control ability, the Pseudomonas bacterium is cultured at a normal culture temperature as a first culture, and then as a second culture, 5 to 5 A method for producing a microbial pesticide, which ensures the number of viable microorganisms and preserves the storage stability, wherein the microorganism is cultured at a low temperature of 15 ° C., dried and formulated using the cultured microorganism.   The method for producing a microbial pesticide according to claim 1, wherein a normal culture temperature in the first culture is 20 to 40 ° C.   The method for producing a microbial pesticide according to claim 1 or 2, wherein the culture time of the first culture is 1 to 50 hours, and the culture time of the second culture is 1 to 50 hours.   The method for producing a microbial pesticide according to any one of claims 1 to 3, wherein the culture microorganism is dried by freeze vacuum drying or pulse combustion drying. 5. The genus Pseudomonas, which is a microorganism capable of controlling agricultural chemicals, is Pseudomonas azotoformans W-14-1 strain (Pseudomonas azotoformans NITE BP-02371). A method for producing a microbial pesticide according to claim 1.