KR101499692B1 - Culture method of spore for Entomopathogenic fungus using natural zeolite ceramic ball, and method of harvesting the same - Google Patents

Abstract

The present invention provides a method for producing spores by culturing using a natural zeolite cerealball of an insect pathogenic fungus having a biological control function and a method for recovering spores obtained by the method, and more particularly, The insect pathogenic fungi using a solid medium for a natural zeolite ceramic ball carrier containing sodium chloride as a starch, an inorganic component and a spore inducing regulator which serve as a carbon source and a role of stably intercalating rice bran extract and natural zeolite ceramic balls as an organic component are cultured , It is possible to efficiently produce a sufficient amount of spores necessary for formulation, and the spores recovered by the method of separating spores produced by the above method are excellent as insecticides for controlling various insects.

Description

TECHNICAL FIELD The present invention relates to a method for producing spores of insect pathogenic fungi using a natural zeolite ceramic ball and a method for separating spores from natural zeolite ceramic balls,

The present invention relates to a method for producing fungal spores using a solid medium for a natural zeolite ceramic ball carrier of an entomopathogenic fungi capable of biologically controllable fungi, a spore separation method using a milling cutter and a spore collection method using a cyclone method About

Recently, the problem of agricultural ecosystem pollution caused by excessive use of pesticides has become a social concern, and interest in agricultural products safety has been increasing both domestically and externally. In Korea, OECD countries have the highest use of pesticide per agricultural land (Kim, YK, 2011. Development on the Plants of Solanaceae (Pepper, Eggplant, Tomato).

Replacing some of the currently used chemical pesticides with microbial pesticides is expected to significantly reduce chemical pesticide use. Bacteria and fungi are commonly used in pest control, and pathogenic fungi used to control powdery mildew include Beauveria , Isaria , Lecanicillium , Aschersonia spp. Various kinds of paper are known. Of these, Beauveria bassiana is an effective antiseptic for major agricultural pests such as greenhouse powder Microbial pesticides with B. bassiana as effective microorganisms are registered. However, the use of fungi in insect pathogenic fungi is difficult and the effect on environment is very different. Therefore, it is necessary to carry out studies that can improve the utilization in the field because it is a very limited use in farmers (Faria, M., Wraight, SP, 2001. Biological control of Bemisiatabaci with fungi., Crop Protect., 20, 767-778.).

Despite the possibility of spore production by insect pathogenic fungi in liquid culture, spores produced in liquid culture are hydrophilic and not easily formulated in oil. Injection spores are also produced in liquid fermentation, but are also hydrophilic and lose their viability relatively quickly during storage. These problems are solved when insect pathogenic fungi are used as biomaterials and there is a demand for technologies capable of mass production of durable spores at low cost (Silman, RW, Nelson, TC, and Bothast, Rj (1991) methods for production of Colletotrichum truncatum spore for use as a mycoherbicide. FEMS Microbiol. Lett. 79, 69-74.).

Solid cultures have been extensively studied in the past when producing fungus spores. Solid cultures such as rice, barley and corn, and solid components derived from grains such as wheat bran have been used. However, In addition, it is difficult to control aseptic as well as to control the environment such as temperature, moisture and pH during cultivation. In addition, the separation of these insoluble solid components and spores is very difficult, and the incubation time is long and the culture cost is high.

Solid culture method of strains using grains Although it is possible to produce spores with high viability and durability, unnecessary insoluble medium components that are not used as nutrients in the culture medium are included in all the processing steps, which lowers the production efficiency and increases production and distribution costs There is also. There arises a problem of dispersion stability during hydration resulting from a large amount of insoluble matter, a problem of spray nozzle passing, and the like.

Accordingly, an object of the present invention is to provide a method for producing a spore of insect pathogenic fungi by a solid culture method using a natural zeolite ceramic ball, So that it can be used as a raw material for a biological control agent by an efficient separation method.

The above object of the present invention is achieved by providing a solid culture method using a natural zeolite ceramic ball of an insect pathogenic fungus and then providing a method of efficiently separating spores produced.

More particularly, the present invention relates to a method for producing insect pathogenic fungal spores, Investigating insect pathogenic fungal spore growth in the type of ceramic balls; Investigating the productivity of insect pathogenic fungus spores in the types of ceramic balls; Examining insect pathogenic fungus spore productivity and spore inducing regulators according to the composition of the deadlock material; And inspecting the insect pathogenic fungal spore content recovered and recovered.

The present invention has an excellent effect of providing a method of efficiently producing an insect pathogenic fungus spore and a method of efficiently recovering spores produced in the above manner.

FIG. 1 is a graph showing the results of investigation of spore productivity in a solid medium for each insect pathogenic fungi according to the present invention. FIG.
FIG. 2 is a photograph showing growth results in various ceramic balls of the insect pathogenic fungi of the present invention, that is, synthetic zeolite, natural zeolite, and natural activated carbon.
FIG. 3 is a graph showing the results of investigation of the spore productivity in various ceramic balls of the insect pathogenic fungi of the present invention, that is, synthetic zeolite, natural zeolite, and natural activated carbon.
FIG. 4 is a graph showing the results of investigation of the spore productivity according to the extract of rice bran in a deadly material containing a spore suspension of a natural zeolite ceramic ball solid medium of an insect pathogenic fungus of the present invention.
FIG. 5 is a graph showing the results of the investigation of the pH spore induction factor in the natural zeolite ceramic ball solid medium of the insect pathogenic fungi of the present invention.
FIG. 6 is a graph showing the result of investigation of the spore induction factor according to the salt concentration in the natural zeolite ceramic ball solid medium of the insect pathogenic fungus of the present invention.
FIG. 7 is a cross-sectional view of a physical spore-recovering device for recovering only the cells and spores cut by the cyclone aspiration method after cutting the cells and spores attached to the surface of the natural zeolite ceramic balls of the present invention with a milling cutter.

The present invention relates to a method of solidifying natural zeolite ceramic balls for the isolation of insoluble matter in a medium during the production of spores of insect pathogenic fungi, and more particularly, to a method for culturing a solid zeolite ceramic ball for the isolation of insoluble pathogenic fungi from microbial extracts and spore suspensions into natural zeolite ceramic balls To produce an insect pathogenic fungus spore, and to the spore isolation by sponge separation by a milling cutter and the spore recovery by the cyclone method.

In the present invention, the natural zeolite ceramic balls are used for adsorbing inorganic additives having a sustained release function of metal elements and phosphates, and for imparting easy-to-handle physical properties, and also to prevent the mixture of insoluble matters in the medium from occurring when spores are separated.

The starch in the present invention is designed in view of the function of the rice bran extract as a crosslinking agent capable of evenly interlocking with the surface of natural zeolite ceramic balls and as a source of carbon source during the growth of insect pathogenic fungi, A suspension is added. The addition rate can be adjusted by adding 1: 11: 3.2 by weight of rice bran extract: starch: spore suspension to weight ratio.

On the other hand, the added inorganic salt in the present invention is sodium chloride, which is developed from the viewpoint that it exists as a mechanism of an inorganic component source and a spore inducing regulator, and the pH control given when culturing by the above culture method is also viewed from the same viewpoint .

The insect pathogenic fungus spores produced by the above-mentioned cultivation method of the present invention are obtained by cutting the cells and spores attached to the surface of the natural zeolite ceramic balls with a milling cutter by a physical machine called a spore separation and recovery machine, The natural zeolite ceramic balls used after spore recovery are provided as a reusable material for cultivation.

Hereinafter, the structure of the present invention will be described in more detail with reference to preferred embodiments, and these examples are only for illustrating the present invention specifically, and the scope of the present invention is not limited by these examples.

Hereinafter, the present invention will be described in more detail with reference to examples.

Example  1 insect pathogenic fungus cereal star In solid medium  Spore productivity survey

100 g each of rice, barley, rice husk, and rice bran were washed three times each as a solid medium of the present invention, immersed in water for 30 minutes, fixed at a moisture content of 40% for 40 minutes, and sterilized at 121 캜 for 1 hour. Solid culture was carried out in a 28 ° C incubator for 15 days after inoculation with a pathogenic fungus of insect pathogenic fungi (7.5 / 1L bottle) in a rice bran extract medium (rice bran: water = 1: 8) for 3 days. After completion of culture, a certain amount of sample was sampled from each medium and diluted with the same dilution ratio, and the content of the spores was examined using a haematocytometer.

The productivity of spore by solid medium was the highest in the rice medium. It is believed that this is due to the increased water retention due to the larger and smaller surface area of the grain compared with other grains. The spore productivity after the final culture was in the order of rice gruel, barley, rice, and rice (Fig. 1).

Example  2 Insect pathogenic fungi  Ceramic Ball ( Synthetic zeolite , Natural zeolite , Natural activated carbon)

We investigated the possibility of solid culture using insecticide hospital molds. A ceramic ball composed of synthetic zeolite, natural zeolite, and activated carbon was cleaned and sterilized at 121 ° C for 30 minutes. The prepared ceramic balls were spread on gauzed patridish, sprayed with a rice bran extract medium containing insect pathogenic fungus, and then sealed and incubated for 3 days in a constant temperature and humidity chamber (28 ° C, 40%).

As a result of cultivation, it was confirmed that insect pathogenic fungi can grow on the surface of the ceramic balls in all three species (Fig. 2).

Example  3 Insect pathogenic fungi  Ceramic Ball ( Synthetic zeolite , Natural zeolite , Natural activated carbon)

It was confirmed that the insecticide fungus could be cultured by using a matrix and the spore productivity of each carrier was investigated. 100 g of each ceramic ball was washed and sterilized at 121 ° C for 30 minutes. After the mixture was treated with the ceramic balls, the prepared clogs containing the prepared spore suspension (3 mL of rice bran extract + 33 g of starch + 7.5 mL of spore suspension) After a certain amount of the gauze was spread in a Patri dish, it was incubated for 12 days in a constant temperature and humidity chamber (28 ° C, 40%).

As a result of spore productivity, natural zeolite ceramic balls showed the best spore productivity in the ceramic ball treatment, followed by natural activated carbon ceramic balls and synthetic zeolite ceramic balls. Especially, spore formation of natural ceramic balls was found to be similar to that of synthetic zeolite ceramic balls. The difference in spore productivity of the synthetic zeolite ceramic balls was changed to alkaline due to the change of the synthetic material due to the high temperature (over 800 ℃) during firing, so that the insect pathogenic fungi did not grow well.

The spore productivity by cereals and ceramic balls was investigated. The spore productivity of natural zeolite ceramic balls was much lower than that of cereals (Fig. 3).

Example  4 Insect pathogenic fungi Natural zeolite Ceramic Ball Solid  In a deadlocked material containing spore suspension On rice bran extract  Spore productivity survey

Spore productivity of insect pathogenic fungi was investigated by culturing on a natural zeolite ceramic ball matrix solid medium for the source of carbon and nitrogen source. 100 g of natural zeolite ceramic balls were cleaned and sterilized at 121 ° C for 30 minutes and then treated with a ceramic sponge suspension containing the prepared spore suspension. Followed by incubation for 12 days in a constant temperature and humidity chamber (28 ° C, 40%).

The spore productivity of insect pathogenic fungi according to the composition of the deadlock material was found to be the highest in the order of Composition 1 (3 mL of rice bran extract + 33 g of starch and 7 mL of spore suspension). These results suggest that as the source of carbon source, as well as the raw rice extract, starch also becomes a secondary source, and its nutritional components become abundant and the mycelial growth progresses and the number of spores decreases. It is common for all fungal spore induction conditions to be produced under extreme conditions (Fig. 4).

Example  5 Insect pathogenic fungi Natural zeolite Ceramic Ball In solid medium  Spore induction Regulatory factor  Research

Generally, one of the growth characteristics of all fungi is to regulate spore induction by changing the external environment (nutrient depletion, physical impact damage, chemical change, temperature, light, humidity). The regulatory factors were investigated in order to increase spore productivity through spore induction in the natural zeolite cera bigball solid medium of insect pathogenic fungi. 100 g of natural zeolite ceramic balls were washed and sterilized at 121 ° C for 30 minutes, and the prepared dead matter (3 mL of rice bran suspension + 33 g of starch + 7.5 mL of spore suspension) was mixed and treated with each ceramic ball After a certain amount of the gauze was spread in a Patri dish, it was incubated for 12 days in a constant temperature and humidity chamber (28 ° C, 40%).

As a result, the spore productivity was different at pH 3 compared to other treatments. As a result, the spore productivity was different at 5% salt concentration of sodium chloride as the other factors. 5 and 6).

Example  6 Insect pathogenic fungi Natural zeolite Ceramic Ball In solid medium  Investigation of separation method of produced spores

2 kg of natural zeolite ceramic balls were washed and sterilized at 121 ° C for 30 minutes. After the mixture was surface-treated with a natural zeolite ceramic ball, the prepared dead material (60 mL of rice bran suspension + 660 g of starch and 150 mL of spore suspension) After spreading in a large glass bowl with gauze, it was incubated for 12 days in a constant temperature and humidity chamber (28 ° C, 40%). After culturing, shade is sufficiently shredded at 30 ° C. Then, the cells and spores attached to the surface of the natural zeolite ceramic balls are cut by a milling cutter by a physical machine called a spore separation and recovering device, and only the cells and spores which are cut by the cyclone aspiration method are recovered. Respectively.

  The concentration of the spores recovered by the physical machine was put into a solution containing the dispersant and mixed thoroughly, and the content of the spores was examined using a haematocytometer. As shown in Table 2, the recovery rate was 63% or more.

Industrial availability

The present invention is an extremely useful invention in the environment-friendly insecticide industry because it has an excellent effect of providing insecticide for pest control using insect pathogenic fungus spores.

Claims (7)

Wherein the insect pathogenic fungi is cultured in a solid medium for a natural zeolite ceramic ball carrier. The method of producing a fungus spore according to claim 1, wherein the solid medium contains an extract of rice bran as an organic component. The method according to claim 2, wherein starch is further contained in the raw rice bran extract and the natural zeolite ceramic balls as organic components of the solid medium. The method of claim 1, wherein the solid medium comprises 1 to 5% sodium chloride as an inorganic component and a spore inducing regulator. The method of claim 1, wherein the solid medium is characterized by a growth condition at pH 3. A method for recovering fungus spores recovered by a cyclone method after separating the fungus spores produced by the method of any one of claims 1 to 5 with a milling cutter.
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