KR100756196B1 - Culture method for producing of high thermal tolerant enthomopathogenic fungal spores - Google Patents

Culture method for producing of high thermal tolerant enthomopathogenic fungal spores Download PDF

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KR100756196B1
KR100756196B1 KR1020060114934A KR20060114934A KR100756196B1 KR 100756196 B1 KR100756196 B1 KR 100756196B1 KR 1020060114934 A KR1020060114934 A KR 1020060114934A KR 20060114934 A KR20060114934 A KR 20060114934A KR 100756196 B1 KR100756196 B1 KR 100756196B1
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김재수
이한영
정봉진
제연호
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주식회사 동부하이텍
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Abstract

A culture method of highly thermal tolerant enthomopathogenic fungi is provided to maintain activity of enthomopathogenic fungi spore stably during circulation period, so that microbial pesticides having high pathogen-controlling activity are produced. A highly thermal tolerant enthomopathogenic fungi are cultured in a solid medium containing corn greach and/or an inorganic salt selected from sodium chloride and potassium chloride or vegetable oil selected from olive oil, corn oil, cotton seed oil, linoleic acid and oleic acid, wherein the amount of inorganic salt or vegetable oil is 0.1-5 wt.% based on the weight of the corn greach; and the enthomopathogenic fungi is Paecilomyces fumosoroseus SFP-198(KCTC 0499BP).

Description

열안정성이 높은 곤충병원성 곰팡이 포자의 배양방법{Culture Method for Producing of High Thermal Tolerant Enthomopathogenic Fungal Spores}Culture Method for Producing of High Thermal Tolerant Enthomopathogenic Fungal Spores

도 1은 본 발명의 실시를 위한 고체배지 종류별 P. fumosoroseus 포자 생산성을 보여주는 그래프이다.1 is a graph showing the productivity of P. fumosoroseus spores according to the type of solid medium for the practice of the present invention.

도 2는 본 발명의 실시를 위한 고체배지 종류별 P. fumosoroseus 포자의 상온 안정성을 보여주는 그래프이다.Figure 2 is a graph showing the room temperature stability of P. fumosoroseus spores by type of solid medium for the practice of the present invention.

도 3은 본 발명의 실시를 위한 옥수수 조분쇄물(corn greach) 고체배지의 첨가배지 종류별 포자생산성 및 열 안정성을 보여주는 그래프이다.Figure 3 is a graph showing the spore productivity and thermal stability of each type of addition medium of corn greach solid medium for the practice of the present invention.

도 4는 본 발명의 실시를 위한 옥수수 조분쇄물(corn greach) 고체배지의 첨가배지로서 무기염 함량별 포자의 열 안정성 및 생산성을 보여주는 그래프이다.Figure 4 is a graph showing the thermal stability and productivity of spores by inorganic salt content as an addition medium of corn greach solid medium for the practice of the present invention.

도 5은 본 발명의 실시를 위한 옥수수 조분쇄물(corn greach) 고체배지의 첨가배지로서 식물성 오일 함량별 포자의 열 안정성 및 생산성을 보여주는 그래프이다.5 is a graph showing the thermal stability and productivity of spores by vegetable oil content as an addition medium of corn greach solid medium for the practice of the present invention.

본 발명은 미생물 농약의 소재로 이용 가능한 곤충병원성 곰팡이(Entomopathogenic fungi)의 열안정성이 높은 포자 배양방법에 관한 것이다.The present invention relates to a method of culturing spores with high thermal stability of entomopathogenic fungi that can be used as a material for microbial pesticides.

최근 화학 살충제의 과다한 사용에 의한 대상 곤충의 내성 증가, 생태계 혼란, 인체에 대한 악영향의 우려 등의 문제점이 줄기차게 제기되어왔다. 이에 대한 대안으로 자연계에 존재하는 곤충병원성 미생물을 이용한 미생물농약의 개발에 대한 관심이 높아지고 있다.Recently, problems such as increased resistance of target insects due to excessive use of chemical insecticides, ecosystem disruption, and concern about adverse effects on the human body have been raised. As an alternative to this, there is a growing interest in the development of microbial pesticides using insect pathogenic microorganisms present in nature.

미생물을 포함하는 생물농약이란 미생물 자체를 직접 이용하거나 미생물을 포함한 제제를 이용하여 농작물에 해를 주는 곤충, 응애, 선충 등의 해충과 각종 식물병원균 또는 잡초를 효과적으로 방제하는데 쓰이는 생물제제라고 할 수 있다. 이러한 생물농약은 유기합성 농약에 비해 저독성으로 약해가 적으며 생태계에 영향이 적고 약제에 대한 내성이나 저항성 유발을 나타내지 않는 등의 많은 장점을 가지고 있음에도 불구하고, 일반적으로 방제 스펙트럼이 제한적이라는 소재 상의 난점과 상품화 과정에서 곰팡이 포자가 고열에 노출되었을 경우 현저한 활성저하가 유발되는 유통과정상의 난점 등으로 인하여 성공적인 제품 개발에는 한계가 있었다.Biopesticides containing microorganisms are biological agents used to effectively control insects, mites and nematodes and various phytopathogens or weeds that harm crops by directly using microorganisms or by using microorganisms. . Although these biopesticides have many advantages such as low toxicity and low toxicity compared to organic synthetic pesticides, less impact on the ecosystem, and no resistance or drug resistance, they generally have a limited control spectrum. In the process of commercialization, mold spores have been limited to successful product development due to the difficulty in distribution, which causes significant deactivation when exposed to high temperatures.

특히 선발된 곤충병원성 미생물이 그 자체로는 방제적 활성을 가진다고 하더 라도 실용화를 위해서는 장기간의 유통기간통안 안정적인 포자활성을 유지하는 것이 관건이다. 이러한 측면에서 제품 보관 중 열에 대한 안정성이 높은 포자의 배양방법의 개발이 시급하게 요구되는 것이다.In particular, even if the selected insect pathogenic microorganisms have control activities in themselves, it is important to maintain stable spore activity over a long shelf life for practical use. In this respect, the development of a method of culturing spores with high heat stability during product storage is urgently required.

본 발명은 상기와 같은 요구에 의해 안출된 것으로서, 본 발명의 목적은 미생물 농약의 소재로 이용가능한 곤충병원성 곰팡이의 열안정성이 높은 포자 배양방법을 제공하는 것이다. The present invention has been made in accordance with the above-described demands, and an object of the present invention is to provide a method of culturing spores with high heat stability of an insect pathogenic fungus that can be used as a material for microbial pesticides.

이러한 본 발명은 곤충병원성 곰팜이를 고체배양하는 과정에서 배지종류별로 포자 내부의 안정성 향상 유도물질의 생산성이 다를 수 있다는 가정하에 1차적으로 곡물배지를 선발하고, 선발된 곡물배지를 보다 개선하기 위해 첨가배지를 선발하는 일련의 연구를 통하여 완성되었다.The present invention primarily selects the grain medium under the assumption that the productivity of the improved inducing material in the spores may be different depending on the type of medium in the process of solid culture of insect pathogenic Gom-Palm, and to further improve the selected grain medium This was completed through a series of studies to select additional medium.

이와 같은 목적을 달성하기 위한 본 발명은 열안정성이 높은 곤충병원성 곰팡이 포자의 배양방법에 관한 것으로, 보다 상세하게는 곤충병원성 곰팡이를 옥수수 조분쇄물(corn greach)을 주재로 하는 고체배지에서 배양하는 것을 특징으로 하는 열안정성이 높은 곤충병원성 곰팡이의 배양방법에 관한 것이다.The present invention for achieving the above object relates to a method for culturing insect pathogenic fungi spores with high heat stability, and more particularly to cultivating the insect pathogenic fungus in a solid medium based on corn greach (corn greach) The present invention relates to a method for culturing insectogenic fungi having high heat stability.

본 발명의 상기 고체배지는 무기염 또는 식물성 오일 중 선택된 1종 이상을 추가로 첨가한 첨가배지일 수 있다.The solid medium of the present invention may be an additional medium further added one or more selected from inorganic salts or vegetable oils.

본 발명에서의 무기염의 첨가는 배지의 수분포텐셜을 낮춤으로서 배양기간동안 수분에 의한 영향을 줄일 수 있다는 견지에서 착안된 것이다.The addition of the inorganic salt in the present invention is conceived in view of reducing the influence of water during the culture period by lowering the water potential of the medium.

한편, 본 발명에서의 식물성 오일의 식물 또는 동물들이 불량환경에서 체내의 지방함량, 특히 불포화지방산 함량을 높여 적응하는 기작이 존재한다는 견지에서 착안된 것이다.On the other hand, it is conceived in view of the mechanism that the vegetable oil or plants of the present invention adapt to increase the fat content, especially unsaturated fatty acid content of the body in a poor environment.

본 발명의 상기 첨가배지의 무기염은 염화나트륨(NaCl) 또는 염화칼륨(KCl) 중에서 선택될 수 있으며, 바람직하기로는 상기 무기염은 염화칼륨(KCl)이 첨가될 수 있다.The inorganic salt of the addition medium of the present invention may be selected from sodium chloride (NaCl) or potassium chloride (KCl), preferably the inorganic salt may be added potassium chloride (KCl).

본 발명의 상기 첨가배지의 식물성 오일은 옥수수 오일(corn oil), 올리브 오일(olive oil), 면실 오일(cotton seed oil) 및 리놀렌산(linoleic acid) 중에서 선택될 수 있으며, 바람직하기로는 올리브 오일(olive oil)이 첨가될 수 있다.Vegetable oil of the additive medium of the present invention may be selected from corn oil (olive oil), olive oil (olive oil), cotton seed oil (cotton seed oil) and linoleic acid (linoleic acid), preferably olive oil (olive) oil) may be added.

또한, 본 발명의 상기 첨가배지는 옥수수 조분쇄물 대비 무기염 또는 식물성 오일을 0.1 내지 10.0 중량%로 첨가하여 조성될 수 있으며, 바람직하기로는 상기 무기염 또는 식물성 오일은 1 중량%로 첨가되어 조성될 수 있다.In addition, the addition medium of the present invention may be formed by adding an inorganic salt or vegetable oil to 0.1 to 10.0% by weight compared to the coarse corn, preferably, the inorganic salt or vegetable oil is added to 1% by weight of the composition Can be.

한편, 본 발명에서 사용한 곰팡이는 대표적인 곤충병원성 곰팡이인 Paecilomyces fumosoroseus SFP-198 균주(이명 코드: DBB2032) (수탁번호: KCTC 0499BP, 균주 특허:특2000-0025982)를 이용하여 열안정성이 높은 포자 생산 방법을 검정하였다.On the other hand, the fungus used in the present invention is a representative insect pathogenic fungi Paecilomyces The fumosoroseus SFP-198 strain (nickname code: DBB2032) (Accession No .: KCTC 0499BP, Strain Patent: Special 2000-0025982) was used to assay a method of producing high heat stability spores.

이하, 본 발명의 구성을 바람직한 실시예를 통하여 보다 상세히 설명할 것이 나, 이들 실시예는 오로지 본 발명을 구체적으로 예시하기 위한 것으로서 본 발명의 범위가 이들 실시예에 의해 제한되는 것은 아니며, 본 발명의 범위는 오직 특허청구범위에 기재된 바에 의해 한정되어야 할 것이다.Hereinafter, the configuration of the present invention will be described in more detail with reference to preferred embodiments, but these examples are only for illustrating the present invention specifically, the scope of the present invention is not limited by these examples, the present invention Is intended to be limited only by the terms of the claims.

<실시예 1> 고체배지 종류별 포자 생산성 및 상온 안정성 비교Example 1 Comparison of Spore Productivity and Room Temperature Stability by Type of Solid Medium

콩(적두), 콩(백두), 쌀, 옥수수, 옥수수(조분쇄물) 100g을 각각 1L 배양병에 넣은 후 121℃ 조건에서 30분 동안 멸균하였다. 고체배지 100g당 40㎖의 증류수를 첨가하여 수분조절을 하였다. SDA+Y broth(Saubraud dextrose 배지+0.5%중량 Yeast extract 첨가)에서 3일간 액체배양한 P. fumosoroseus SFP-198종균을 1㎖/병으로 접종한 후, 27℃ 인큐베이터에서 3주간 고체배양을 진행하였다. 배양 완료 후 각각의 배지에서 생산된 포자만 채(sieve)로 수집하여 바로 포자발아율을 조사한 다음, 10일 후 포자발아율을 조사하여 포자 안정성을 비교하였다. 동시에 haemacytometer를 사용하여 포자수 함량을 조사하였다.100 g of soybeans (red beans), soybeans (baekdu), rice, corn, and maize (crude grind) were put into 1L culture bottles and sterilized for 30 minutes at 121 ° C. Water control was performed by adding 40 ml of distilled water per 100 g of solid medium. P. fumosoroseus cultured in SDA + Y broth (Saubraud dextrose medium + 0.5% weight yeast extract) for 3 days After inoculating SFP-198 spawn at 1 ml / bottle, solid culture was performed for 3 weeks in a 27 ° C. incubator. After completion of the culture, only spores produced in each medium were collected into sieves (sieve), and the spore germination rate was immediately examined. After 10 days, the spore germination rate was examined to compare spore stability. At the same time, the spore count was examined using a haemacytometer.

고체배지 종류별 포자 생산성 및 상온 안정성을 조사한 결과, 곡물배지 중에서는 옥수수 조분쇄물(corn greach) 조건에서 가장 높은 포자 생산성을 보였다. Corn greach는 배지 표면적이 크고 옥수수 자체를 분쇄시킴으로써 수분 보유력이 높아졌기 때문에 상대적으로 많은 양의 포자를 생산한 것으로 판단되었다 (도 1). 배양 종료 후 수확한 포자의 상온안정성은 corn greach 배지 > 콩(백태) > 콩(적두) = 쌀배지 순으로 corn greach 배지에서 가장 안정성이 높은 포자가 생산되었다 (도 2). 포자 생산성 및 안정성 측면에서 corn greach가 가장 적합한 고체배지인 것으로 판단되었다.As a result of investigating the spore productivity and room temperature stability according to the types of solid media, the highest spore productivity was obtained in the grain grease condition of corn medium. Corn greach was determined to produce a relatively large amount of spores because of the medium surface area and the increased water retention by grinding the corn itself (Fig. 1). The room temperature stability of the spores harvested after the end of the culture was produced in the most stable spores in corn greach medium in order of corn greach medium> soybeans (beans)> soybeans (red) = rice medium (Fig. 2). Corn greach was considered to be the most suitable solid medium in terms of spore productivity and stability.

<실시예 2> Corn greach 고체배지의 첨가배지 종류별 포자 생산성 및 열안정성 비교Example 2 Comparison of Spore Productivity and Thermal Stability of Various Types of Corn Greach Solid Medium

옥수수(조분쇄물) 10g과 KCl, NaCl, Sucrose, Sorbitol, Dextrin, 탈지유(Skim milk), 폴리에틸렌 글리콜(PEG), 폴리크로필렌 글리콜(PPG), Linoleic acid, Corn oil, Olive oil에서 선택된 1종 0.1g 그리고 물 4㎖을 시험관에 넣은 후 121℃ 조건에서 30 분 동안 멸균하였다. SDA+Y broth에서 3일간 액체배양한 P. fumosoroseus SFP-198종균을 0.1㎖/시험관으로 접종한 후, 27℃ 인큐베이터에서 3주간 고체배양을 진행하였다. 배양 완료 후 담체를 동결건조 후 포자만 붓으로 수집하였다. 포자체를 50℃ 조건에서 2시간동안 열처리한 후 포자발아율을 조사하였다. 동시에 haemacytometer를 사용하여 포자수 함량을 조사하였다.10 g of maize (crude), one selected from KCl, NaCl, Sucrose, Sorbitol, Dextrin, skim milk, polyethylene glycol (PEG), polycropropylene glycol (PPG), Linoleic acid, Corn oil, Olive oil 0.1 g and 4 ml of water were placed in a test tube and sterilized at 121 ° C. for 30 minutes. P. fumosoroseus cultured in SDA + Y broth for 3 days SFP-198 seedlings were inoculated with 0.1 ml / vial, followed by solid culture for 3 weeks in a 27 ° C. incubator. After completion of the culture, the carrier was lyophilized and only spores were collected by brush. After spores were heat treated at 50 ° C. for 2 hours, the spore germination rate was examined. At the same time, the spore count was examined using a haemacytometer.

Corn greach 고체배지의 첨가배지 종류별 포자 생산성 및 열안정성을 조사한 결과, Corn greach 첨가배지 중에서 50℃의 열처리 조건에서 비열처리 조건과 유사한 포자발아율을 보인 첨가물은 KCl, Linoleic acid, Corn oil, Olive oil로 확인되었으며 이중에서 Olive oil의 효과가 가장 우수하였다. 포자 생산성은 KCl, NaCl, Linoleic acid, Olive oil 조건에서 무처리와 유사한 수준을 보였다. 하지만 나머지 배지조건에서는 포자생산성이 무처리보다 감소하는 결과를 보였다 (도 3). As a result of investigating the spore productivity and thermal stability by the type of medium added to the corn greach solid medium, the additives showing similar spore germination rate under the heat treatment condition of 50 ℃ were KCl, Linoleic acid, Corn oil and Olive oil. Among them, the effect of Olive oil was the best. Spore productivity was similar to that of no treatment under KCl, NaCl, Linoleic acid and Olive oil. However, the remaining media conditions showed a decrease in spore productivity than no treatment (Fig. 3).

<실시예 3> Corn greach 고체배지의 첨가배지 함량별 포자 생산성 및 열안정성 비교Example 3 Comparison of Spore Productivity and Thermal Stability of Corn Greach Solid Medium

옥수수(조분쇄물) 10g과 NaCl, KCl, Olive oil, Corn oil, Cotton seed oil을 각각 0.1, 0.2, 0.4, 0.8g 그리고 물 4㎖을 시험관에 넣은 후 121℃ 조건에서 30분 동안 멸균하였다. SDA+Y broth에서 3일간 액체배양한 P. fumosoroseus SFP-198를 0.1㎖/시험관으로 접종한 후, 27℃ 인큐베이터에서 3주간 고체배양을 진행하였다. 배양 완료 후 담체를 동결건조 후 포자만 붓으로 수집하였다. 포자체를 50℃ 조건에서 2시간동안 열처리한 후 포자발아율을 조사하였다. 동시에 haemacytometer를 사용하여 포자수 함량을 조사하였다.10 g of corn (crude), NaCl, KCl, olive oil, corn oil, and cotton seed oil, 0.1, 0.2, 0.4, 0.8 g and 4 ml of water, respectively, were placed in a test tube and sterilized at 121 ° C. for 30 minutes. P. fumosoroseus cultured in SDA + Y broth for 3 days After inoculating SFP-198 with 0.1 ml / vial, solid culture was performed for 3 weeks in a 27 ° C. incubator. After completion of the culture, the carrier was lyophilized and only spores were collected by brush. After spores were heat treated at 50 ° C. for 2 hours, the spore germination rate was examined. At the same time, the spore count was examined using a haemacytometer.

무기염으로 KCl과 NaCl을 검토한 결과, 첨가량이 증가할수록 포자의 열안정성은 다소 증가하는 경향을 보이기는 하지만, 오히려 포자 생산성은 감소하는 결과를 보였다. 2가지 무기염 중에서는 NaCl보다는 KCl이 포자 생산성 및 열 안정성 향상에 도움이 되는 것으로 확인되었으며, 최종적으로 KCl 1% 내외가 적합한 첨가량으로 판단되었다 (도 4). 첨가배지로서 식물성 오일 3종을 검토한 결과, 마찬가지로 첨가량이 증가할수록 포자의 열안정성은 다소 증가하는 경향을 보이기는 하지만, 오히려 포자 생산성은 감소하는 결과를 보였다. 포자의 열안정성 측면에서는 olive oil이 가장 우수하였으며 포자 생산성을 고려하여 olive oil 1% 내외를 적합한 첨가량으로 선발하였다 (도 5).As a result of examining KCl and NaCl as inorganic salts, the thermal stability of spores tended to increase slightly as the amount of addition increased, but the spore productivity decreased. Among the two inorganic salts, KCl rather than NaCl was found to help improve spore productivity and thermal stability, and finally, about 1% of KCl was determined to be a suitable addition amount (FIG. 4). As a result of examining three vegetable oils as the added medium, the thermal stability of the spores tended to increase slightly as the amount of the added oil increased, but the spore productivity decreased. Olive oil was the best in terms of thermal stability of the spores, and in consideration of spore productivity, olive oil was selected at an appropriate amount of about 1% (Fig. 5).

이상에서 상술한 바와 같이 본 발명에 따른 곤충병원성 곰팡이 포자의 배양방법은 포자 생산성이 우수하면서 열안정성이 높은 효과적인 배양방법인 것으로 평가되었다.As described above, the culturing method of the insect pathogenic fungus spores according to the present invention was evaluated to be an effective culturing method having excellent spore productivity and high thermal stability.

또한, 본 발명의 따른 곤충병원성 곰팡이 포자의 배양방법은 방제적 활성이 높은 미생물농약을 생산하는데 효과적으로 적용될 수 있을 것으로 기대되었다.In addition, it was expected that the method for culturing insect pathogenic fungus spores according to the present invention could be effectively applied to produce microbial pesticides having high controllability.

Claims (5)

곤충병원성 곰팡이를 옥수수 조분쇄물(corn greach)을 주재로 하는 고체배지에서 배양하는 것을 특징으로 하는 열안정성이 높은 곤충병원성 곰팡이의 배양방법.A method of culturing insectogenic pathogenic fungi, characterized by culturing insect pathogenic fungi in a solid medium based on corn greach. 제1항에 있어서, 상기 고체배지는 무기염 또는 식물성 오일 중 선택된 1종 이상을 추가로 첨가한 첨가배지인 것을 특징으로 하는 열안정성이 높은 곤충병원성 곰팡이의 배양방법.The method of claim 1, wherein the solid medium is an cultivation method of insecticidal fungus having high heat stability, characterized in that the addition medium further added at least one selected from inorganic salts or vegetable oils. 제2항에 있어서, 추가로 첨가되는 무기염은 염화나트륨(NaCl) 또는 염화칼륨(KCl) 중에서 선택되는 것을 특징으로 하는 열안정성이 높은 곤충병원성 곰팡이의 배양방법.The method of culturing a highly thermostable insectogenic fungus according to claim 2, wherein the additional inorganic salt is selected from sodium chloride (NaCl) or potassium chloride (KCl). 제2항에 있어서, 추가로 첨가되는 식물성 오일은 올리브 오일(olive oil), 옥수수 오일(corn oil), 면실 오일(cotton seed oil), 리놀렌산(linoleic acid), 올레익산(oleic acid) 중에서 선택되는 것을 특징으로 하는 열안정성이 높은 곤충 병원성 곰팡이의 배양방법.The method of claim 2, wherein the additional vegetable oil is selected from olive oil (corn oil), corn oil (corn oil), cotton seed oil (cotton seed oil), linoleic acid, oleic acid (oleic acid) A method of culturing insect pathogenic fungi having high thermal stability. 제2항 내지 제4항 중 어느 한 항에 있어서, 상기 첨가배지는 옥수수 조분쇄물 대비 무기염 또는 식물성 오일을 0.1 ~ 5 중량%로 첨가되어 조성되는 것을 특징으로 하는 열안정성이 높은 곤충병원성 곰팡이의 배양방법.According to any one of claims 2 to 4, wherein the addition medium is a high heat-stable insect pathogenic fungi, characterized in that the composition is added to the inorganic salt or vegetable oil to the corn coarse to 0.1 to 5% by weight. Cultivation method of.
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