KR101425046B1 - Mass production method of Paecilomyces lilacinus HY―４ - Google Patents

Abstract

The present invention relates to a method for mass production of Paecilomyces fungi and a method for mass production thereof, and more particularly, to a method for mass production of Paecilomyces lilacinus strain HY-4 in an economical industrial scale To a method for producing industrial spores of a filamentomyces fungus for industrial application. The mass production medium, culture method and spore production method of the fungus belonging to the genus Faecalomyces according to the present invention can be useful for industrialization for controlling various pests.

Description

Mass production method of Paecilomyces lilacinus HY-4}

The present invention relates to a mass production method of my process (Paecilomyces) sp indeed L, more particularly L saengse will indeed My process Sinners (Paecilomyces The present invention relates to a culture medium for industrially mass-producing lilacinus HY-4 for use in pest control, a culture method using the culture medium, and a method for producing industrial spores of Fasylomyces fungi using the culture method.

The domestic and overseas agricultural industry in the 21st century is expected to have faster and more quantitative and qualitative growth than ever before, as the food safety is emphasized along with the stable food supply and well-being demands due to abnormal weather. Especially, the problem of safe food is related to the primary safety of the crops produced. Therefore, the government is planning to establish an environmentally-friendly agricultural development plan. In order to increase the proportion of production of environmentally friendly agricultural products and to reduce the amount of chemical pesticides and fertilizer We are pursuing.

The development of biopesticides that can replace organic synthetic chemical pesticides as a solution for human and environmental toxicity problems and eco-friendly agriculture is proposed as the most promising alternative. In the United States, extensive research and development is underway in recognition of the pesticide research in the field of green chemistry.

Currently, there are more than 370 registered biologically active pesticides in the world, of which about 270% are pesticides, and the development of bio-pesticides is mainly carried out by small companies. The development of microbial pesticides based on microorganisms is a major feature. Microbial pesticides The most commonly used microbes are fungi and bacteria, followed by pest control.

In order to control pests, in Korea, much effort has been made to control green grass powder, cabbage moth, larvae, and brown planthopper, as well as beetle larvae, which are applied to golf course grass and ginseng field. However, And the problems of environmental pollution caused by such problems. Therefore, there is an increasing need for environmentally friendly pest control in order to minimize damage caused by soil and water pollution caused by chemical insecticides used in the past, toxicity to human body, harmful endocrine disruptor, appearance of resistant insect pests, and reduction of useful natural enemies.

In order to control these pests eco-friendly, they have been using natural methods for decades, but now many farmers are using BT and natural products that are sold domestically. However, most of these formulations are imported from China and other countries, but the development of resistance is rapid, resulting in short life cycle of developed products.

Therefore, it is urgently required to find new effective microorganisms having excellent control effect for the development of insecticidal insect pests and soil pest control agents that can not be controlled by the existing BT and natural materials. Among them, especially insect pathogenic fungi are effective in controlling and have been already developed and used as products in foreign countries. The main ingredient of insect pathogenic fungi products is Beauveria bassiana or Metarhizium anisopliae . However, these products are probiotic products using fungus spores, and it is estimated that the market expansion is sluggish due to the problems of mass production and application of products.

Insect Pests Pest control using fungi is an environmentally friendly means of controlling, selectively acting only on target insects, inhibiting the appearance of resistance to pests, allowing continuous control, and no toxicity to the environment, It is possible to produce insect pathogenic fungi suitable for the domestic environment in a wide range of countries. Therefore, it is a practical practice to isolate, mass-produce and formulate fungi suitable for the domestic environment.

Even in Korea, the research and development to date have been limited to the development of insect pathogenic fungi probiotics, making it difficult to achieve industrial achievements. As a result, it is difficult to secure the economical efficiency of the actual developed products and the excellent effects in the field, and these problems are important factors in the sluggish market expansion.

The global biopharmaceutical market continues to grow, and in 2005, the pesticide market is forming a 2.5% market. Currently, it is expected to expand to 4.3% of the total pesticide market based on the rapid expansion of demand for eco-friendly agricultural products. A report by The New Biopesticide Market in January 2006 predicts that pesticides will grow at a rate of 1.1 per cent compared to annual growth of 9.9 per cent. The market is expected to expand further when bio-pesticides are developed to replace chemical pesticides.

In Korea, the bio-pesticide market is not large compared to chemical pesticides, but it is expected that the market will expand due to the expansion of eco-friendly agriculture. In particular, the government plans to reduce the use of chemical pesticides by 40% The market is expected to expand further when developed.

Since 2000, despite the government's efforts to support the first eco-friendly agriculture, the use of domestic chemical pesticides has not decreased at all, and the market for environmentally friendly pesticides has also been less than 3%. The main reasons for this are lack of directionality (strategy) and lack of marketable product development technology for the lack of R & D and commercialization of developed technologies.

Research on bio-pesticides including microbial pesticides and biochemical pesticides in Korea started sporadically in universities and government research institutes from the late 1970s, but since the 1990s, research and development has been activated to a certain extent. In 1997, And the government is supporting the eco-friendly agriculture to reduce the usage of chemical pesticides, so the development of bio-pesticides and microbial agents is proceeding in earnest.

In the case of research and development of biological pesticides and microbial agents, early research and development focused on securing excellent microorganisms or active substances derived from new microorganisms mainly in laboratory experiments. Recently, venture companies have focused on mass production and formulation technologies It is accumulating.

In addition, the history of research and development of domestic microbial pesticides is very long, but market entry is very limited due to price and activity problems. Due to social demands, production of safe food and consideration of the environment have been increased, and due to technological progress, a considerable number of pesticides have been used as insecticides, but they still have a substantial dependency on chemical pesticides.

However, due to lack of development strategy and know-how, it still remains in the early stage of technology development, and mass production and formulation technology closely related to industrialization is still not enough to make industrial products.

As a result of intense efforts for the development of industrial microbial pesticides, the present inventors have previously isolated and identified a novel strain of the genus Faecalis maize, which can effectively control a heating insect pest having a high market demand (US Pat. No. 7,435,411; -0475131 strain Accession No. KCTC 0395BP).

Accordingly, the present inventors have made intensive efforts to economically and industrially mass-produce the strain of the genus Faecalomyces in the above-mentioned inventions. As a result, they have found that the optimal culture medium for the mass production of the strain, the culture environment, And confirming the method of producing industrial spores of the genus Mytilus.

It is an object of the present invention to provide an optimum medium composition of a strain of the genus Faecilomyces established for industrial mass production for utilizing Paecilomyces sp. Strain for pest control, And a method for mass production of spore masses of Fasylomyces fungi using such a culture method.

In order to achieve the above object, the present invention provides a medium composition for mass production of molds of Paecilomyces that can control pests damaging various commercially important agricultural crops.

In addition, the present invention provides a method for mass production for mass production of industrially and economically large quantities of molds of the genus Faecalomyces.

The present invention also provides a method for mass production of spores capable of mass-producing industrially and economically large molds of the genus Faecalomyces.

Specifically, the present invention provides

1) pre-culturing the faecal matter of Mycophthaeae;

2) liquid culture of pre-cultured Fasylomyces fungi in a primary culture; And

3) a step of solid-culturing the liquid cultured filamentomyces fungi by a secondary culture.

In addition,

1) pre-culturing the beetle of the genus Faisilomyces;

2) liquid culture of pre-cultured Fasylomyces fungi in a primary culture;

3) solid culturing the liquid culture of the filamentomyces fungus by a secondary culture; And

4) isolating the spores from the solid-cultured medium. The present invention also provides methods for producing spores of the genus Faecalomyces.

In addition, the present invention relates to a method for producing a microorganism belonging to the genus Lactobacillus, comprising the step of cultivating a microorganism belonging to the genus Lactobacillus, comprising a pupa having 0.5 to 1.0% (w / v) of the total medium composition as a nitrogen source and 0.5 to 1.0% (w / v) Provide a culture medium for the fungus.

The present invention establishes an optimal medium composition for the mass production of Paecilomyces fungi and confirms that it can mass-produce the fungus belonging to the genus Faecilomyces , which can control the soil pest, Can be useful for industrialization for the control of various soil pests harming various important agricultural crops.

FIG. 1 is a diagram showing the results of growing the strains of the Fabilomyces lysinus HY-4 in a culture medium using Czapek medium, Czapek pupa, rice, and wheat bran respectively.
FIG. 2 is a graph showing the results of cultivating and growing recovered spores of Fasilomyces lysinus HY-4 in Czapek medium, Czapek medium containing pupa, rice medium, and wheat bran, And the number of viable cells after the incubation for a minute was measured.
Fig. 3 shows the stability of the recovered spore after culturing in the medium using the Czapek medium, the culture medium containing the pupa, the rice medium, and the wheat bran, respectively, with the strains HY-4 And the results are shown in FIG.
4 is a graph showing the germination percentage (%) over time (18 h and 36 h) for various temperatures (24 ° C, 28 ° C, 32 ° C and 36 ° C) of the Fasylomyces lysinaceus HY-4 strain.
FIG. 5 is a graph showing the growth rate (μm / h) per hour at various temperatures (24 ° C., 28 ° C., 32 ° C. and 36 ° C.) of the Fasylomyces lysinaceus HY-4 strain up to 116 hours.
FIG. 6 is a graph showing the rate of survival to the spotted mite according to the treatment with the spiny mosaic rythianus HY-4 spore suspension and the number of spotted mite (B) dead due to the fungal disease symptom.

Hereinafter, the present invention will be described in detail.

The present invention provides a medium for mass production of Paecilomyces fungi.

My process (Paecilomyces) in the mold chamber is L will My process chamber to deposit the L accession No. KCTC 0395BP Sinners (Paecilomyceslilacinus) which is one preferred HY-4 is not limited to this.

The culture medium according to the present invention is preferably a PDA (Potato Dextrose Agar) medium, a PDB (Potato Dextrose Broth) medium, or a czapek medium as a basic medium, but is not limited thereto and may be a medium for culturing fungi Are all usable.

The medium according to the present invention is preferably prepared by mixing a carbon source, a nitrogen source or an inorganic material with water, and the medium is preferably sterilized by high pressure, but is not limited thereto.

As the carbon source, it is preferable to use glucose, corn starch, saccharose, molasses, wheat bran, rice bran or the like, more preferably bran or rice bran, but is not limited thereto.

As the nitrogen source, pupa, yeast extract, soybean meal, corn steep liquor, malt extract, peptone and the like are preferably used, and pupae are more preferably used, but not limited thereto.

It is preferable to use potassium chloride (KCl), magnesium sulfonate (MgSO ₄ ), ferrous sulfate (FeSO ₄ ), sodium nitrate (NaNO ₃ ), potassium phosphate (K ₂ HPO ₄ ) But is not limited thereto.

The culture medium according to the present invention preferably contains a pupa of 0.5 to 1.0% (w / v) of the total medium composition as a nitrogen source, more preferably a pupa of 0.5% (w / v) But is not limited thereto.

The culture medium according to the present invention preferably contains 0.5-1.0% (w / v) of rice or wheat bran as a nitrogen source, preferably 0.5% (w / v) But is not limited thereto.

When the rice according to the present invention is used as a nitrogen source, it is preferable to use a supernatant after sterilization by mixing water and rice at a ratio of 1: 1. If wheat bran is used, the ratio of bran to water is adjusted to 4: But it is not limited thereto.

In one embodiment of the present invention, Paecilomyces lilacinus strain HY-4 was used as a culturing microorganism for mass production, and the microorganism was used as an insect pest and a pest After isolating microorganisms having insecticidal effect against the insect pests from the soil samples inhabited, Paecilomyces was identified as Paecilomyces , and named as Paecilomyces lilacinus HY-4. It is a microorganism deposited with the Institute of Genetic Engineering (Accession No. KCTC 0395BP).

In one embodiment of the present invention, in order to optimize the production of bacterial cells of the Pseudomaeias lirasinus HY-4 strain, a culture medium in which 0.5% (w / v) pupa was ground with a nitrogen source under the existing Czapek medium was used And the spore concentration was measured after culturing the strain. As a result, it was confirmed that the addition of 0.5% pupa at the same culture condition increased spore production of about 1.78 times (see Table 1).

In one embodiment of the present invention, in order to establish a medium composition for industrial mass production of Pseudomaeias lirasinus strain HY-4, rice and wheat bran are selected as an optimal nitrogen source which is easy to supply and economical, The spore concentration and viable cell count were measured in a medium containing 0.5% (w / v) rice or wheat bran respectively. As a result, it was confirmed that when cultured with rice and bran, the spore concentration and viable cell number were increased in comparison with the case of containing the pupa in Czapek (see FIG. 1 and Table 2).

In one embodiment of the present invention, in order to confirm the stability of the Pseudomaeces lirasinus HY-4 strain at various temperatures, the dried powder of the filamentomyces spores in the medium according to the present invention was cultured at various temperatures, Were measured. As a result, it was confirmed that 98% stability was maintained at 70 ° C. for 15 minutes. In particular, it was confirmed that the stability was maintained at 80 ° C. or higher than that of the other medium when grown in a medium using rice Reference).

In one embodiment of the present invention, in order to confirm the safety of the strain of the Fylulomycetis lilacinus HY-4 against the pH, the Fylulomycetes spore-dried powder in the medium according to the present invention was cultured under various pH conditions, Were measured. As a result, it was confirmed that although the number of spores grown by the medium composition was different, it had stability from pH 5 to pH 10 (see FIG. 3).

In conclusion, the culture medium in which 0.5% (w / v) concentration of rice or bran was added to czapek medium as a basic medium increased the spore concentration and viable cell count of Pesylomycetis lirasinus strain HY-4 , It is confirmed that the stability against temperature and pH is increased, and thus it can be used as a medium for mass production of Pesylomyces lysinus HY-4 strain.

The present invention also provides a method of culturing for the mass production of Paecilomyces fungi.

Specifically, the culturing method is preferably, but not limited to, carried out by a method including the following steps:

1) pre-culturing the faecal matter of Mycophthaeae;

2) liquid culture of pre-cultured Fasylomyces fungi in a primary culture; And

3) a step of solid-culturing the liquid culture of the filamentomyces fungus by a secondary culture.

In the above culture method, it is preferable that the fungus belonging to step 1) is Paecilomyces lilacinus HY-4 deposited with Accession No. KCTC 0395BP, but not limited thereto, All molds are available.

In the above culture method, the pre-culture of step 1) is preferably a PDA (Potato Dextrose Agar) medium as the basic medium, and the liquid culture of step 2) uses PDB (Potato Dextrose Broth) And it is preferable, but not limited, to use czapek medium as the basic medium for the solid culture of step 3).

In the culturing method, the culturing of step 1) to step 3) preferably includes a carbon source or a nitrogen source, but is not limited thereto. As the nitrogen source, pupa, yeast extract, soybean meal, corn steep liquor, malt extract, peptone and the like are preferably used, but the pupa is more preferably used, but not limited thereto. As the carbon source, it is preferable to use glucose, corn starch, saccharose, molasses, wheat bran, rice bran or the like, more preferably bran or rice bran, but is not limited thereto. Here, the pupa is preferably added in an amount of 0.5 to 1.0% (w / v), more preferably 0.5% (w / v), but not limited thereto. Preferably, the rice or wheat bran is added in an amount of 0.5 to 1.0% (w / v), more preferably 0.5% (w / v), based on the whole composition of the medium.

In the above culture method, the culturing of step 1) to step 3) is preferably carried out at 24 ° C to 32 ° C, more preferably 28 ° C to 32 ° C, but is not limited thereto.

In one embodiment of the present invention, the germination rate by temperature was measured in order to confirm the optimal growth temperature of Fasylomyces lysinaceus HY-4. As a result, the germination rate showed a high germination rate at 24 ° C to 32 ° C, and the germination rate was high in the order of 32 ° C, 28 ° C and 24 ° C (see FIG. 4).

In one embodiment of the present invention, the growth rate in diameter (μm / h) of mycelium with respect to the temperature of Fasylomyces lysinaceus HY-4 was measured. As a result, the growth rate per hour was the highest at 32 ° C and was also high at 28 ° C (see FIG. 5).

Therefore, it is preferable that the optimal culturing temperature of the Fabolomyces cerevisiae HY-4 strain is 24 ° C to 32 ° C. When considering the germination rate per hour and the growth rate per hour, the culturing temperature is 32 ° C, Lt; 0 > C or more, it becomes difficult to form spores.

In the above culture method, the pre-culture of step 1) is preferably cultured for 7 to 10 days, more preferably for 7 to 8 days, but is not limited thereto. The liquid culture of step 2) is preferably cultured for 7 to 10 days, more preferably for 7 to 8 days, but not always limited thereto. The culture of step 3) is preferably cultivated for 7 to 25 days, more preferably for 20 to 24 days, but not always limited thereto.

The present invention also provides a method for mass production of spores of Paecilomyces fungi.

Specifically, the spore production method is preferably, but not limited to, performed by a method including the following steps:

1) pre-culturing the fungi of Paecilomyces ;

2) liquid culture of pre-cultured Fasylomyces fungi in a primary culture;

3) solid culturing the liquid culture of the filamentomyces fungus by a secondary culture; And

4) separating spores from the solid cultured medium.

In the above culture method, it is preferable that the fungus belonging to step 1) is Paecilomyces lilacinus HY-4 deposited with Accession No. KCTC 0395BP, but not limited thereto, All molds are available.

In the above culture method, the pre-culture of step 1) is preferably a PDA (Potato Dextrose Agar) medium as the basic medium, and the liquid culture of step 2) uses PDB (Potato Dextrose Broth) And it is preferable, but not limited, to use czapek medium as the basic medium for the solid culture of step 3).

In the culturing method, the culturing of step 1) to step 3) preferably includes a carbon source or a nitrogen source, but is not limited thereto. As the nitrogen sources, pupa, yeast extract, soybean meal, corn steep liquor, malt extract, peptone and the like are preferably used, but the pupa is more preferably used, but not limited thereto. As the carbon source, it is preferable to use glucose, corn starch, saccharose, molasses, wheat bran, rice bran or the like, more preferably bran or rice bran, but is not limited thereto. Here, the pupa is preferably added in an amount of 0.5 to 1.0% (w / v), more preferably 0.5% (w / v), but not limited thereto. Preferably, the rice or wheat bran is added in an amount of 0.5 to 1.0% (w / v), more preferably 0.5% (w / v), based on the whole composition of the medium.

In the above culture method, the culturing of step 1) to step 3) is preferably carried out at 24 ° C to 32 ° C, more preferably 28 ° C to 32 ° C, but is not limited thereto.

In the above culture method, the pre-culture of step 1) is preferably cultured for 7 to 10 days, more preferably for 7 to 8 days, but is not limited thereto. The liquid culture of step 2) is preferably cultured for 7 to 10 days, more preferably for 7 to 8 days, but not always limited thereto. The culture of step 3) is preferably cultivated for 7 to 25 days, more preferably for 20 to 24 days, but not always limited thereto.

In the above culture method, the spore separation in step 4) can be separated through a spore recovery process from a solid culture medium, and such spore collection can be carried out using various methods known in the art.

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

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

< Example  1> Facilomyces Lira sinus HY Of -4 strains Pre-culture

A PDA (Potato Dextrose Agar) medium was used for storage and mass culture of the Fabilomyces lysinus HY-4 strain (Accession No. KCTC 0395BP). The preculture was cultured in Petri dishes (87 X 15 mm) in a PDA medium at 28 ° C for 7 days in a dark room.

< Example  2> Facilomyces  Industrial culture of the genus Escherichia coli

For the cultivation of Fasylomyces lysinus HY-4 strain, the mycelium was maximized in the culture medium by liquid culture in the primary culture, and then the secondary culture, which is a secondary culture for industrial storage and industrial microbial insecticide preparation, was performed.

Specifically, for the mycelial cultivation, the first liquid culture was cultured in PDB medium for 7 days under the conditions of 28 ° C and 180 rpm. Then, the cells were cultured in a Petri dish (150 × 25 mm) under the condition of 25 mL of Czapek culture medium, and 2 mL of mycelial cultured primary cultures were plated on a spreader and cultured for 20 days.

< Example  3> Facilomyces  Industrial bulk recovery method of spores of the genus strain

The spores were recovered through a scraper in a culture medium for the industrial mass recovery of spores of the Fylulomycetes fungus formed through 20 days solid culture in Petri dishes.

< Example  4> Pesylomyces  Medium composition for mass production of genus strains

In order to optimize the production of bacterial cells of the Pesylomyces lysinus HY-4 strain, a culture medium for mass production for the microbial pesticide application was completed using a medium in which the pupa was replaced with a nitrogen source under the existing Czapek medium. An edible product was purchased from the market and dried in a dry oven, followed by pulverization. The spore concentrations in the Czapek medium and the Czapek medium such as those of Example 1, Example 2 and Example 3 were measured in a medium containing 0.5% (w / v) pupa.

As a result, as shown in Table 1, it was confirmed that spore production of about 1.78 times was increased by adding 0.5% pupa in the same culture condition (Table 1).

Substrate Conidia (g) Czapek 2 X 10 ¹⁰ Czapek + 0.5% pupa 7 x 10 ¹⁰

< Example  5> Pesylomyces  Medium composition for industrial mass production of genus strains

Using a pupa, the yield of spore production was increased by 1.78 times in the cultivation of facilomiasis, but it was confirmed that there was a problem in the processing of the pupa and in the supply and pupa of the pupa. In case of pupae, seasonal influences and problems of supply and demand are problematic. Especially, it seems that there is a difficulty in economical efficiency with microbial insecticides for the purpose of producing a large amount of nitrogen source. Rice and bran were selected as nitrogen sources. As a medium, rice was used as a supernatant after sterilization at a ratio of water to rice of 1: 1. Bran was used for sterilization after adjusting the ratio of bran to water to 4: 1. The spore concentration and viable cell counts in the medium containing 0.5% (w / v) rice or bran in the Czapek medium and Czapek medium as described in <Example 1>, <Example 2> and <Example 3> Respectively. In a medium containing 0.5% (w / v) pupa, rice, and wheat bran respectively in Czapek medium and Czapek medium as described in <Example 1>, <Example 2> and < Seth spores were collected and the yield, spore concentration and number of live cells were measured and compared.

As a result, as shown in Fig. 1 and Table 2, it was preferable that the spore formation number and the viable cell count contained the pupa in Czapek, but considering economical efficiency and industriality, the culture using rice and bran was more efficient (Fig. 1 and Table 2).

Medium composition Yield
(g / plate) Spore concentration
(conidia / g) Viable cell count
(cfu / g) Czapek 0.03 3.2 X 10 ¹⁰ 6.6 X 10 ⁹ Czapek + Pupa 0.12 5.7 X 10 ¹⁰ 1.2 X 10 ¹⁰ rice 50-60 4.0 X 10 ⁹ 8.3 X 10 ⁸ bran 25-30 2.5 X 10 ¹⁰ 8.6 X 10 ⁹

< Example  6> Facilomyces  Identification of the stability of the genus Escherichia coli at various temperatures

To confirm the stability of Peyeromyces lysineus HY-4 strain at various temperatures, the dried powder of Phellinus lysineus sp. Cultured in different media was placed in a test tube (0.1 - 0.2 g / tube) After incubation for 5, 10, 15, 20 and 30 minutes in an oven (JEIO TECH, FO-600M) at 70 ° C. and 80 ° C., spores were distributed in a concentration of 10 ⁴ (conidia / mL) through a hemocytometer , Diluted to 0.1% (v / v) tween 80, and then 200 μL was cultured in PDA medium at 60 ° C., 70 ° C. and 80 ° C. for 24 hours as in Example 2 and Example 3 Germination rate was measured.

As a result, as shown in Fig. 2, it was confirmed that the sample was allowed to stand at 70 占 폚 for 15 minutes to have a stability of 98%. However, it was confirmed that the stability was decreased after 10 minutes at 80 ° C. In particular, it was confirmed that the growth rate of the strain of the genus Faecilomyces grown in the medium using rice was lower than that of the other medium but the basic stability at 80 ° C was still maintained (FIG. 2).

< Example  7> Facilomyces  Variety of genus strains pH Stability results for

In order to confirm the safety of various strains of Phellinus lysinaceus HY-4 against various pHs, dried powder of Phellinus linteus sp. Cultured in different media was placed in a test tube (0.1 - 0.2 g / tube) (V / v) tween 80, and 200 μL was added to the PDA medium in the same manner as in Example 2, except that the concentration of the spores was adjusted to 10 ⁴ (conidia / mL) And < Example 3 > were measured for germination after culturing for 24 hours.

As a result, as shown in FIG. 3, it was confirmed that although the number of spores grown by the medium composition was different, it had stability from pH 5 to pH 10 (FIG. 3).

< Example  8> Facilomyces  Investigation of optimum culture temperature of strains

To determine the optimal growth temperature of Fasilomyces lysinaceus HY-4, the germination rate at each temperature was measured. Culture temperatures were 24 ℃, 28 ℃, 32 ℃ and 36 ℃, respectively. (1 × 10 ⁵ conidial / mL) of Fasylomyces lysinus HY-4 was diluted with the PDA medium and 0.1% (v / v) Tween-80, and 100 μL was added thereto by the method of Example 3 Culturing was carried out at different culture temperatures in the same manner as in < Example 2 &gt;. Three groups were grown and germination was measured electron microscopically through 10 pools of each sample at random.

As a result, as shown in FIG. 4, it was confirmed that germination was observed at 36 hours except for 36 ° C. as a result of measurement at 18 hours and 36 hours. The germination rate was shown in the order of 32 ° C, 28 ° C and 24 ° C in the order of 18 hours (Fig. 4).

The growth rate in μm / h of mycelium was also measured at 116 hours.

As a result, as shown in FIG. 5, the growth rate per hour from 116 hours was 105.2 μm / h at 32 ° C. and was higher than 79.3 μm / h at 28 ° C. (FIG.

Therefore, it was confirmed that the spore formation becomes difficult when the temperature is above 36 ° C, and the optimal culture temperature is from 24 ° C to 32 ° C, and more preferably, when the germination rate per hour and the growth rate per hour are considered, Respectively.

< Example  9> Facilomyces  Genus mold insecticide Hydrating agent  Produce

A wettable powder of the powder that is hydrated when diluted with water is prepared for the purpose of stably preparing the raw material of the strain Fasilomyces lysinus HY-4. Specifically, a spore (1.1 × 10 ⁹ conidiavirusible conidia / gram) 10 (100 μg / ml) recovered as in Example 3 was inoculated with the medium of Example 5 after inoculation with the strain of the present invention, % (w / w) and 90% (w / w) of an excipient were uniformly mixed to prepare a wettable powder.

< Example  10> Fanny Shilomyces  Genus mold insecticide Granulation  Produce

A granule which can be used as a granular form was prepared for the purpose of stable formulation of the raw material of the strain Lysineus HY-4. Specifically, a spore (1.6 × 10 ⁹ visible conidia / gram) recovered as in Example 3, including the medium of Example 5, was inoculated with the strain of the present invention, 10% (w / w) and 90% (w / w) of excipient were uniformly mixed to prepare a granule.

< Example  11> Fanny Shilomyces  Manufacture of mold insecticide emulsion

An emulsion which can be used as a granule as a granule for the purpose of stable formulation of the raw material of the strain Fasilomyces lysineus HY-4 was prepared. Specifically, a spore (1.6 × 10 ⁹ visible conidia / gram) recovered as in Example 3, including the medium of Example 5, was inoculated with the strain of the present invention, 10% (w / w) and 90% (w / w) excipients were used. A variety of vegetable oils and mineral oils may be used as excipients to increase the stability of spores. As the vegetable oil, soybean oil, corn oil, sunflower oil, olive oil and the like can be used. As the mineral oil, kerosene, paraffin oil and the like can be used.

< Example  12> Securing insects

Tetranychus as a heating pest urticae , two-spotted spider mite) were used as insects. The spotted mite is a heating insect pest which has a rapid resistance to chemical acaricide and a rapid generation number of 10 ~ 11 times per year, which causes a great damage to all the cut flowers and caterpillars. It is a problematic pest around the world because of the rapid generation of generations and rapid resistance to chemical pesticides. Spotted mite is easy to find host because it has wide host range. Kidney bean, which is used as a typical nursery, is easier to use because it has a larger leaf area than other hosts. They were kept at 25 ℃, 70% relative humidity and 16L: 8D.

< Example  13> Bioassay by spraying method

Immersion method in which the insect itself is immersed in a fungus spore suspension, and spray method on a fungus spore suspension on a target insect. Among the spraying methods mainly used for testing the insects, the insect pest set in the bioassay is a pseudomonas, It was judged to be unsuitable for the resultant assay, and the biological assay method using the spray was determined by the biological assay method of the present invention.

< Example  14> Bioassay by spraying with industrial cultured spores

A 20 mm diameter round bean curd was placed on a 90 mm dish and a 15 ml spotted mite adult was placed on it. Then 1 mL of a spore suspension of the previously prepared Fylulomyces lysineus HY-4 strain (1 X 10 ⁷ conidia) was sprayed using the method of Example 13 to perform bioassay. This procedure was repeated three times.

As a result, as shown in FIG. 6, a mortality rate exceeding 70% was observed in the strain of the Fabilomyces lysinaceus HY-4, and fungal diseases were also found in the hatchlings (FIG. 6).

In addition, the <Example 13> and the like, based on the information of the active spores formulated strain in substantially the bioassay ^{1 mL (1 X 10 7 conidia} ) falling active spores per area (cm per ^second) during when spraying the spore suspension .

As a result, as shown in Table 3, the spiny mite treated with Lactinosyl Lysineus HY-4 showed a higher mortality rate than the untreated control group despite the fact that the number of actually treated active spores was low, And most of them showed mold symptoms (Table 3). Therefore, it was confirmed that Pseudomyshesriaca sinensis HY-4 biologically controls the mucosal irritation.

Medium composition Cm ² active conidia rice 2577 bran 483

The present invention relates to a method for the treatment of molds of the genus Paecilomyces , in particular Paecilomyces &lt; RTI ID = 0.0 &gt; lilacinus HY-4 strain and spores thereof, and thus can be usefully used for industrialization for controlling various pests.

Korea Biotechnology Research Institute KCTC0395BP 19971027

Claims (12)

1) pre-culturing Paecilomyces lilacinus HY-4 fungus deposited with accession number KCTC0395BP in a PDA (Potato Dextrose Agar) medium as a primary culture medium;
2) The Paecilomyces lilacinus HY-4 fungus pre-cultured was used as a liquid basal medium in a PDB (Potato Dextrose Broth) medium to which a pupa was added at 0.5 to 1.0% (w / v) Culturing in a medium to obtain a large amount of mycelium; And
3) The step of culturing the mycelium obtained in step 2) as a solid basic medium in a medium supplemented with 0.5 to 1.0% (w / v) of pupa, rice or wheat bran as a whole in czapek medium A method for mass production of Paecilomyces lilacinus HY-4 mold spores, comprising:
delete delete The method of mass production of Paecilomyces lilacinus HY-4 mold spores according to claim 1, wherein the medium of step 1) to step 3) comprises a carbon source or a nitrogen source.
delete delete delete The method for mass production of Paecilomyces lilacinus HY-4 mold spores according to claim 1, wherein the culture of step 1) to step 3) is cultivated at 28 ° C to 32 ° C.
The method according to claim 1, wherein the culture of step 1) or step 2) is cultivated for 7 to 10 days, and the culture of step 3) is cultivated for 15 to 25 days ( Paecilomyces lysinaceus lilacinus ) HY-4 Method for mass production of mold spores.
A method for mass production of Paecilomyces lilacinus HY-4 fungal spores according to claim 1, further comprising the step of separating spores from the solid cultured medium.
delete delete

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