KR20060047000A - Method for preparing conidia of ampelomyces quisqualis, formulation method comprising the conidia and the composition thereof - Google Patents

Abstract

The present invention provides a method for producing spores of Ampelomyces quisqualis having a biological control function, a method for preparing a formulation including the spores, and a composition prepared by the method. The present invention can be used as an effective microbial control method by separating and collecting spores of Amfelomyces quisqualis as a microbial control agent that maintains improved hydration and increased activity.

Ampelomyces quisqualis, spores

Description

Method for preparing conidia of Ampelomyces quisqualis, formulation method comprising the conidia and the composition according to a method for producing spores of Ampelomyces quiqualis, and a method for preparing a formulation comprising the spores

1 is a comparison of the medium prepared by using the present invention and the medium prepared by the conventional method, 1: in the photo, conventional method, 2: barley flour luxury supernatant of the present invention, 3: soybean meal dissolved supernatant of the present invention, 4: According to the present invention, 5: distilled water shows that the turbidity of the insoluble and insoluble precipitate and the turbidity of the medium are remarkably improved compared to the results of the conventional method.

Figure 2 is a comparison picture of Ampelomyces quisqualis culture cultured using the present invention and the culture cultured by the conventional method, the left side of the photograph of the conventional method, the right side shows the method of the present invention Insoluble and insoluble substances in the interior are markedly removed as compared to the conventional method.

3 is a photograph of the release of conidia from pycnidia.

Figure 4 is a 500- and 1000-time scanning electron microscope (SEM) photographs of Ampelomyces quisqualis spores (conidia) isolated using the present invention.

Figure 5 is a 20,000-time scanning electron microscope (SEM) photograph of Ampelomyces quisqualis spores (conidia) isolated using the present invention.

The present invention relates to the field of biological control against plant pathogens, and more particularly, to a plant pathogen control agent using Ampelomyces quisqualis , and is suitable for spore separation collection of Ampelomyces quisqualis . It relates to a microbial culture method, spore separation method, formulation method as a biological control agent.

As the side effects of gunpowder pesticides are pointed out, interest in environmentally friendly biological control continues to increase. Currently, selective control of plant pathogens using various antagonists is being researched and developed worldwide. Examples of strains used as biological control agents include Bacillus thuringiensis , Verticillium lecanii , Autographica californica NPV, Beauvaria bassiana , and Bacillis. Bacilis subtilis , Pseudomonas fluorescens , Streptomyces griseoviridis , Trichoderma harzianum and Ampelomyces qualquisquilis ).

Ampelomyces genus overwinters in the form of pycnidia in nature (Yarwood, 1939), isolated from many types of powdery mildew (Shin, 1994), and is known to be host-free. (Blumer, 1993; Clare, 1964). Ampelomyces quisqualis is a double parasitic bacterium that invades mycelia and spores of powdery mildew bacteria and forms sickness in them. This suppresses the spore germination of powdery mildew bacteria and kills powdery mildew bacteria. Using this mechanism of action, the discovery and formulation of new strains of Ampelomyses quisqualis for powdery mildew control have been conducted in various countries.

In the late 1980s, ampelomyses quisqualis was formulated in Austria, but high humidity was required to germinate spores, reducing the control against powdery mildew (Benlanger et al., 1998).

U.S. Patent No. 5,190,745 (1993) proposes a new strain, Ampelomyces quisqualis 10, and a method for controlling powdery mildew using the same, which is 'AQ10'. Registered for.

Korean Patent No. P-0332480 (1999) 'New Ampelomyses quisqualis 94013 and the control of phytopathogens using the same' proposes a method for isolation and culture of strain AQ94013 showing more effective control than AQ10.

The formulation of biological control agents is largely classified into suspension and dry powder. Suspension is applied to phytopathogens by introducing microorganisms having biological control ability into water or oil, and then applying them to phytopathogens. In detail, suspension forms detailed forms of emulsion, suspension concentrate and encapsulated suspension. Can be divided into: Dried powder is applied directly to phytopathogens in powder form. It can be divided into dust, granule and brequettes (in order of increasing particle size) according to the particle size. (wettable powder) can also be included in the form of dry powder (Rhodes, 1993).

It is an object of the present invention to develop a control agent having increased hydration (solubility) and activity as a biological control agent than a conventional product in the form of a hydrating agent.

Existing products are filtered through the entire culture of Ampelomyses quisqualis cultured with barley flour and soybean meal as nutrients to remove the liquid components passed through, adding an extender (lactose, etc.), lyophilizing, pulverizing and interfacing. It was a product in the form of a hydrate, which went through a series of processes such as adding an active agent. This filtrate contains all the insoluble components of barley flour and soybean meal, insoluble mycelium components, and insoluble metabolites used as a medium. Together they showed a problem of sinking to the bottom of the container. It also has the problem that, from an economic point of view, unnecessary insoluble media ingredients that are not used as nutrients are included in all process steps, reducing production efficiency and increasing production and distribution costs. In order to overcome the problems of dispersion stability when hydrated from a large amount of insoluble materials, and problems of passing through a spray nozzle, a crushing process is performed to reduce the particle size after freeze-drying. However, this conventional method causes physical stress on the spores and causes the spores to be inhibited. In order to solve this problem, there is a need for an economical and practical technique for easily removing the insoluble components in the medium production step to facilitate the separation and collection of ampelomyses quisqualis spores after incubation.

It is an object of the present invention to provide a control agent in the form of a hydrating agent which has an increased hydration power (solubility) and activity as a biological control agent than a conventional product.

That is, the present invention is to provide an efficient biological control formulation that can overcome the above problems by using Ampelomyces quisqualis strain.

To this end, it is intended to provide a mass culture method of microorganisms for spore separation. In addition, it is intended to provide a method for separating Amphelomyces quisqualis spores (conidia) from the culture.

In addition, it is to provide a formulation method of high spore activity and hydration using the separated spores.

In order to achieve the above object, the present invention comprises the steps of (a) removing insoluble material from the grains before incubation; (b) inoculating the medium in which the insoluble substance has been removed by inoculating Ampelomyces quisqualis; (c) filtering the Ampelomyces quiqualis culture; And (d) milling the filtered Amphelomyces quisqualis mycelium and the bottle agglomerate to crush the bottle agglomerate.

As a preferred example, the separation method of the present invention is (a) adding water to the carbon source, followed by gelatinization by heating and centrifugation, and adding water to the nitrogen source, dissolving at room temperature and then centrifuging the supernatant containing water-soluble nutrients. Obtaining; (b) mixing the carbon source supernatant and the nitrogen source supernatant, and inoculating the supernatant liquid mixture by inoculating Ampelomyces quisqualis; (c) filtering the Ampelomyces quiqualis culture; And (d) milling the filtered Amphelomyces quisqualis mycelium and the bottle agglomerate.

In another aspect, the present invention comprises the step of removing the water by centrifugation of the spore separation solution separated by the spore separation method, adding an extender to the spores from which the water is removed, and lyophilizing the spore composition to which the extender is added. It provides a method for producing phytopathogen control formulation.

In the present invention, the carbon source is preferably barley, rice, millet, brown rice, or sorghum, and the nitrogen source is preferably soybean meal.

In addition, the gelatinization process of the present invention is characterized in that it further comprises adding a salt, the gelatinization process is preferably carried out under alkaline conditions.

In another aspect, the present invention provides a composition for controlling phytopathogens prepared by a method for producing a phytopathogen control formulation.

Hereinafter, the present invention will be described in detail.                     

Existing Ampelomyces quisqualis cultivation studies have been mostly optimization optimization for obtaining a large amount of spores through the change of conditions, such as medium type, culture environment, the present invention is to provide a culture method for spores separate collection do.

Barley, rice, millet, brown rice, sorghum, etc. may be used as the carbon source, and soybean meal was selected as the nitrogen source. Conventional methods were put into the grain and soybean meal used as a carbon source in water at the same time and heat sterilized at 121 ℃ and then inoculated with the bacteria. This method, due to the various insoluble substances contained in the grains, agglomeration between Ampelomyces quusqualis bacteria and insoluble components occurs during culture, making it difficult to separate spores after culture. Therefore, the present invention provides a condition that can be easily separated before culturing Ampelomysis quisquales bacteria by removing the insoluble material that interferes with the spore separation process in grains before the culture. To this end, the grains and soybean meal used as carbon sources are separated and various insoluble substances such as crude fibers are removed, and only starch, protein and other water-soluble nutrients are taken as raw materials for culture.

In the case of grains such as barley flour, which is used as a carbon source, starch particles in the grains are dispersed in water and gelatinized by heating to obtain starch, which is a major nutritional substance. In this process, salts such as FeSO ₄ , CaCl ₂ , and CaCO ₃ may be added to promote the growth of Ampelomyces quiqualis. Gelatinization of starch is promoted when alkali conditions or some salts (KOH, KI, etc.) are added, so NaOH may be added to reduce the temperature and time (but sulfates rather impede gelatinization, so Fefel ₄ is amphelomycin). Useful for growing Seth Quisqualis, but detrimental to luxury). Centrifuge the sonicated solution to obtain a supernatant containing water-soluble nutrients.

In the case of soybean meal as a nitrogen source, unlike soy flour, protein components in soybean meal are denatured during heat treatment, so that the soybean meal is dispersed in water and slowly stirred for several hours under room temperature alkaline conditions to dissolve soybean protein and centrifuged. Take a supernatant containing protein and other water-soluble nutrients. Soy protein is most soluble in pH 9 conditions (Smith et al., 1972).

Each supernatant prepared by the above method is mixed at an appropriate ratio (see FIG. 1), and then adjusted to pH 6-8, which is the growth condition of Ampelomyses quisqualis, and then inoculated with Ampelomysis quisqualis. This is incubated for 7 days under 20 to 30 ° C. under dark and aerobic conditions. Ampelomyses quisqualis cultured in this way is incubated in a state of very low insoluble impurities unlike the conventional method (see Fig. 2).

The present invention seeks to provide a method for obtaining spores from liquid cultured Ampelomyces quisqualis alumni.

In general, mycelium forms spores between the branches of mycelium, and can be easily separated and separated by proper agitation. However, in the case of Ampelomyces quisqualis, conidia are formed in pycnidia. In the resulting form, a suitable method is needed for the discharge of spores from the stinging. In Figure 3 it can be seen that the spores are released from the bottle awake. Only a spores with an average size of 6.9 × 3.0 μm (Lee, 1999) have an average size of 62.8 × 40.5 μm (Lee, 1999), and are not suitable for direct formulation of the bottle angle due to low hydration problems and problems with spray nozzle passage. It should be taken out of the bottle awake and formulated.

Milling is suitable as a method for discharging spores in the bottle shell. Milling can be classified into 'force by collision' and 'force by friction' according to the principle of force used, and the application field is different depending on the purpose of use and the type of sample. In general, the collision is the principle of breaking the crushed particles, which destroys the entire material to be crushed, thereby reducing the particle size. The friction is the principle of reducing the particle size by slicing finely from the surface of the material. Ampelomyses quisqualis need a proper way to explode the spores to release the spores. When a collision force is applied, it is difficult to control the strength of the force and to selectively destroy only the sickness. Ball milling, a typical case of crushing by friction, allows the spores to be ejected gradually by minimizing the stress on the spores, and by grinding the milling time, rotation speed and ball size You can easily adjust the degree. Application of the ball milling method to the Ampelomyces quisqualis culture resulted in efficient splintering and separation of spores without harming the spores. After ball milling, distilled water was sprinkled using a metal sieve to filter out impurities such as pathogen residue and spore separation liquid was obtained, and no physical damage of the spores was found (see FIGS. 4 and 5).                     

The present invention is to provide a formulation of a suitable biological control agent using Ampelomyces quisqualis spores obtained by the above method.

In order to formulate the spores purely separated by the above method in the form of a hydrate, this experiment removes the water from the spore separation liquid by centrifugation, and properly adds a filler, a dispersant, a surfactant, and the like. After blending, lyophilization is carried out. As the extender, lactose, skim milk, etc., as a dispersant, microcrystalline cellulose (MCC), carboxymethyl cellulose (CMC), carrageenan, etc., as a surfactant, polyoxyehylene sorbitan monolaurate (Tween 20), polyoxyehylene sorbitan monostearate (Tween 60) ), polyoxyehylene sorbitan monooleate (Tween 80), nonylphenol, ethoxylated phosphate ester sodium salts, etc., and a small amount of minerals such as silica or calcium chloride (CaCl ₂ ) to prevent aggregation or hardening during production and distribution. (mineral) was added. The additives are hydrated in an appropriate amount of distilled water and then mixed with ampelomyses quisqualis spores, followed by rapid freezing and freeze drying. In the present invention, the spore mixture rapidly frozen at −80 ° C. was pulverized to a size of about 3 mm or less and then lyophilized, unlike the conventional method of undergoing lyophilization immediately after rapid freezing. As it is lyophilized in a predetermined ice state in advance as described above, it is possible to omit the crushing process after drying, thereby reducing the physical stress transmitted to the spores. This method also increases the surface area to facilitate sublimation of water molecules during lyophilization. This is possible by removing the insoluble components that interfere with hydration through pure spore separation, so that the particles can be sufficiently dissolved during hydration even if the particle size is larger than the conventional one. Moreover, even without the addition of dispersants and surfactants showed better hydration than the conventional one.

The present invention will be described in more detail with reference to the following examples, but are not intended to limit the protection scope of the present invention to the following examples.

Gelatinization conditions, extraction conditions, milling and impurity removal methods, etc. mentioned in the following examples are selected only those suitable to illustrate the present invention from the methods known in the art, and the scope of the present invention is limited to the conditions of the following examples It is not limited.

Comparative Example 1

Barley flour (3%) and soybean meal (3%) were suspended in water without pretreatment and used for culture. As a result, insoluble components remain in the culture medium at high density. This causes many problems such as poor hydration in the post process. As shown in FIG. 1, the medium solution prepared by the conventional method shows a very opaque form compared to the development method, and as shown in FIG. 2, insoluble components remain in the culture medium in large quantities.

Example 1

Preparation of Liquid Culture Medium

3% (w / v) of barley flour, 0.05% (w / v) of CaCl ₂ and 0.1% (w / v) of CaCO ₃ are dispersed in distilled water. This was heated at 90 to 110 ° C. for 1 to 10 minutes to gelatinize the starch in barley flour and centrifuged at 3000 to 8000 rpm for 1 to 3 minutes to obtain only the aqueous supernatant.

Soybean meal, a nitrogen source, was dispersed in water at 3% (w / v), stirred for several hours at pH 9, and centrifuged at 3000 to 8000 rpm for 1 to 3 minutes to obtain only an aqueous supernatant from which insoluble matters were removed. However, soy protein extraction conditions are not limited to the above cases.

Each of the supernatants prepared by the above method was mixed at a ratio of 1: 1, and then adjusted to the optimal growth pH of Ampelomyses quisqualis 6.5, and inoculated with 3% (v / v) Ampelomyses quisqualis bacteria. Then, the culture was stirred for 7 to 10 days under a culture temperature of 25 ° C., dark conditions, and aerobic conditions.

Example 2

Separate collection of spores

Ampelomyces quisqualis cultured by the above method was filtered to filter out agglomerates of some metabolites that are much larger than 62.8 × 40.5 μm pycnidia and washed several times with distilled water. Remove other components than the lease. The Ampelomyses quisqualis mycelium and the pathophyllum thus obtained were put in a container together with a ball for milling, and milled at a milling time of 10 minutes, a ball size of 5 mm and a rotational speed of 50 rpm. Pop). After milling, the liquid in the container is filtered by sprinkling with sterile water using a metal body with a diameter of 26 µm. This size is such that spores can pass through, but pathogen debris and other mycelium cannot pass.

Example 3

The spore liquid separated by the method of Example 2 was centrifuged to remove moisture and to obtain spores. As an extender, 100% skim milk (skim milk) by weight of spores removed from water and 5% calcium chloride (CaCl ₂ ) by weight of spores from which water was removed were dispersed in distilled water of the same weight as the dehydrated spores. Mix with spores The blend according to the above method is pulverized after rapid freezing at -80 ° C, and only particles of about 3 mm or less are taken using a 3 mm sieve and then lyophilized for 24 hours. After lyophilization, 10% of the surfactant based on the spore weight from which water is removed may be further mixed.

Overcoming the existing Ampelomyces quisqualis control formulation method including insoluble media components, mycelium, and metabolites discharged as mold grows, only spores are collected and formulated as hydrated microbial pesticides. As a result, it is possible to produce a control agent having improved water solubility (solubility). In addition, the physical stress of the spores can be reduced by performing the grinding process in the frozen state before freeze drying. In addition, by removing and cultivating unnecessary components other than nutrients before cultivation to increase the production efficiency of the incubator, it is possible to reduce the production cost by maintaining a high hydration power without adding a special dispersant and surfactant. The separate collection of spores allows for more efficient concentration of more Ampelomyces quisqualis spores per volume (or weight), thereby reducing distribution costs.

Claims (9)

(a) preliminarily removing insoluble material from the grains prior to incubation; (b) inoculating the medium in which the insoluble substance has been removed by inoculating Ampelomyces quisqualis; (c) filtering the Ampelomyces quiqualis culture; And and (d) milling the filtered Amphelomyces quisqualis mycelium and the bottle agglomerates to break the bottle agglomerates. The method of claim 1 wherein (a) adding water to the grains, which are carbon sources, gelatinizing by heating and centrifuging, adding water to a nitrogen source, dissolving in alkaline conditions at room temperature, and centrifuging to obtain a supernatant containing water-soluble nutrients; (b) mixing the carbon source supernatant and the nitrogen source supernatant, and inoculating the supernatant liquid mixture by inoculating Ampelomyces quisqualis; (c) filtering the Ampelomyces quiqualis culture; And (d) milling the filtered Ampelomyces quisqualis mycelium and the bottle agglomerate to break the bottle agglomerates. (a) centrifuging the spore separation liquid separated in claim 1 or 2 to remove moisture; (b) adding an extender to the dehydrated spores; And (c) lyophilizing the spore composition to which the extender is added. The method of claim 2 wherein the carbon source is barley, rice, millet, brown rice, or sorghum. The method of claim 2, wherein the nitrogen source is soybean meal. The method of claim 2 wherein the gelatinization of step (a) further comprises adding a salt. The method of claim 2, wherein the gelatinization of step (a) is performed under alkaline conditions. The method of claim 6 wherein the salt is FeSO ₄ , CaCl ₂ or CaCO ₃ . A composition for controlling phytopathogenic bacteria prepared by the method of claim 3.

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