KR102650171B1 - Metarhizium pemphigi 111 strain for controlling bulb mite and uses thereof - Google Patents
Metarhizium pemphigi 111 strain for controlling bulb mite and uses thereof Download PDFInfo
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- KR102650171B1 KR102650171B1 KR1020210142675A KR20210142675A KR102650171B1 KR 102650171 B1 KR102650171 B1 KR 102650171B1 KR 1020210142675 A KR1020210142675 A KR 1020210142675A KR 20210142675 A KR20210142675 A KR 20210142675A KR 102650171 B1 KR102650171 B1 KR 102650171B1
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- metarhizium
- mites
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- pemphigi
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Abstract
본 발명은 응애 방제능 및 UV-B 내성을 가지는 메타리지움 펨피기(Metarhizium pemphigi) 111 균주(기탁번호: KACC 83052BP), 상기 균주 또는 이의 배양물을 유효성분으로 포함하는 응애 방제용 조성물 및 상기 조성물을 식물, 식물의 종자 또는 식물 식재 토양에 처리하는 단계를 포함하는 응애 방제 방법에 관한 것이다.The present invention relates to Metarhizium pemphigi 111 strain (Accession Number: KACC 83052BP) having mite control ability and UV-B resistance, a composition for controlling mites comprising the strain or its culture as an active ingredient, and the above It relates to a method for controlling mites comprising applying the composition to plants, plant seeds, or plant planting soil.
Description
본 발명은 뿌리응애 방제능을 가지는 메타리지움 펨피기(Metarhizium pemphigi) 111 균주 및 이의 용도에 관한 것이다.The present invention relates to Metarhizium pemphigi 111 strain having root mite control ability and its use.
식물 생육의 가장 큰 장애 요인으로 지적받고 있는 해충과 식물병을 방제하기 위하여 다양한 작물보호제들이 사용되고 있으며, 더욱 효과적인 보호제의 개발을 위한 연구 개발이 전 세계적으로 활발히 이루어지고 있다. 식량 생산 등의 경제적인 목적 또는 조경을 위한 목적 모두 식물을 키우는 데 있어서 해충의 방제를 위한 작물보호제는 거의 필수적으로 요구되고 있으며, 그에 따른 경제적 비용도 매우 높은 실정이다. 현재까지 이러한 작물보호제는 거의 대부분이 화학물질을 이용하여 개발되고 이용되어 왔으며, 그로 인해 근래에는 이들 화학물질을 바탕으로 한 작물보호제의 인축에 대한 위해성을 비롯하여 생태계 파괴 등의 여러 가지 부작용이 잇따르고 있다. 또한 화학물질을 이용한 살충제의 경우 많은 해충들이 저항성이 발달하여 그 방제를 더욱 어렵게 하고 있으며, 이러한 화학살충제에 대한 해충의 저항성은 연중 많은 세대가 발생하는 흡즙성 미소해충들에서 특히 심하게 발생하고 있다. 이러한 작물보호제의 부작용을 해결하기 위하여 다양한 친환경 방제 방법들이 개발되고 있으며, 그 중 주목받고 있는 방법 중의 한 가지가 곤충병원성(entomopathogenic) 미생물을 이용한 미생물 살충제의 개발과 이용이다.Various crop protection agents are being used to control pests and plant diseases, which are pointed out as the biggest obstacles to plant growth, and research and development to develop more effective protection agents is being actively conducted around the world. Crop protection agents for pest control are almost essential when growing plants for both economic purposes such as food production or landscaping purposes, and the resulting economic costs are also very high. To date, most of these crop protection agents have been developed and used using chemicals, and as a result, in recent years, crop protection agents based on these chemicals have been causing various side effects, including risk to human populations and destruction of the ecosystem. . In addition, in the case of pesticides using chemical substances, many pests have developed resistance, making their control more difficult. Resistance of pests to these chemical pesticides is particularly severe in sucking micropests that generate many generations throughout the year. To solve the side effects of these crop protection agents, various eco-friendly control methods are being developed, and one of the methods that is attracting attention is the development and use of microbial pesticides using entomopathogenic microorganisms.
미생물 살충제의 소재로 이용될 수 있는 미생물은 세균, 바이러스 그리고 곰팡이 등 다양한 미생물이 있으며, 그 중에서 곤충병원성 곰팡이는 가장 오랜 역사를 지니고 있다. 곤충병원성 곰팡이는 곤충에 병을 일으켜 치사시키는 미생물로서 곤충 기주에 침입하여 증식하는 과정 중에 다양한 세포외 효소나 독소물질 그리고 2차 대사산물들을 생산하여 기주 곤충의 면역 반응을 극복하거나, 기주를 치사시키는데 이용되고 있으며 그와 더불어 다른 미생물들과의 경쟁에도 이용되고 있다. 이러한 특징을 이용하여 곤충병원성 곰팡이의 다양한 대사산물에 의한 항세균 및 항진균 활성에 대한 연구 개발 역시 최근에 활발히 이루어지고 있다.There are a variety of microorganisms that can be used as materials for microbial pesticides, including bacteria, viruses, and molds, and among them, entomopathogenic fungi have the longest history. Entomopathogenic fungi are microorganisms that cause disease and death in insects. During the process of invading and proliferating in insect hosts, they produce various extracellular enzymes, toxins, and secondary metabolites to overcome the host insect's immune response or kill the host. It is also used to compete with other microorganisms. Taking advantage of these characteristics, research and development on the antibacterial and antifungal activities of various metabolites of entomopathogenic fungi has also been actively conducted recently.
미생물 살충제 소재로써 가장 많이 이용되는 곤충병원성 곰팡이는 뷰베리아(Beauveria)와 메타리지움(Metarhizium) 속에 속하는 곰팡이들이다. 이들 곰팡이 균주들을 이용하여 나비목 해충이나 딱정벌레목 해충 등 매우 다양한 해충에 대한 살충제가 개발되어 시판되고 있다. 이들 곤충병원성 곰팡이를 이용한 살충제는 살아있는 곰팡이를 소재로하여 개발되기 때문에, 개발된 살충제의 보관, 유통 및 처리시의 안정성이 매우 중요하게 평가되고 있다. 곰팡이의 활성에 영향을 줄 수 있는 여러 가지 환경 인자들 중에서도 특히, 열안정성과 자외선 안정성이 매우 중요한 요소로 여겨지고 있다. 따라서, 최근에는 곰팡이를 이용한 살충제의 개발에 있어서 이들 열안정성과 자외선 안정성이 높은 곰팡이 균주를 이용하려는 연구가 활발히 이루어지고 있다.The entomopathogenic fungi most commonly used as microbial pesticide materials are those belonging to the genera Beauveria and Metarhizium . Using these fungal strains, pesticides against a wide variety of pests, such as lepidopteran pests and coleopteran pests, have been developed and sold on the market. Since pesticides using these entomopathogenic fungi are developed using living fungi, the stability of the developed pesticides during storage, distribution, and processing is evaluated as very important. Among the various environmental factors that can affect the activity of fungi, thermal stability and ultraviolet ray stability are considered very important factors. Therefore, in recent years, research has been actively conducted to use fungal strains with high heat stability and UV stability in the development of pesticides using fungi.
뿌리응애(Rhizoglyphus robini Claparede)는 응애목(Acarina) 가루응애과(Acaridae)에 속하는 해충으로, 토양에 널리 분포하고 있으며, 마늘, 쪽파 등 파속 작물과 백합, 글라디올러스 등의 구근류 14과 28종의 작물에 피해를 준다. 뿌리응애는 토양속에서 뿌리를 물리적으로 가해하여 1차 피해를 입힌 뒤, 가해로 생긴 상처로 곰팡이나 세균의 침입이 이루어지면서 부패, 병원균의 감염 등 2차 피해를 유발한다. 뿌리응애를 방제하기 위해서는 비펜트린(bifenthrin), 델타메트린(deltamethrin), 프로메캅(promecarb), 디아지논(diazinon) 등의 합성농약이 주로 사용되고 있으나, 뿌리응애의 저항성 획득 및 환경오염 문제 등이 문제점으로 나타나고 있어 그 방제가 매우 어려운 해충으로 여겨지고 있다.Root mite ( Rhizoglyphus robini Claparede) is a pest belonging to the order Acarina and family Acaridae . It is widely distributed in the soil, and affects 28 species of crops in 14 families and bulbs such as allium crops such as garlic and chives, as well as lilies and gladiolus. It causes damage. Root mites cause primary damage by physically damaging roots in the soil, and then fungi or bacteria invade through wounds caused by the damage, causing secondary damage such as rot and infection with pathogens. To control root mites, synthetic pesticides such as bifenthrin, deltamethrin, promecarb, and diazinon are mainly used, but there are problems such as acquisition of resistance by root mites and environmental pollution. Due to this problem, it is considered a pest that is very difficult to control.
한편, 한국공개특허 제2016-0140560호에는 '해충방제 및 항균활성을 가지는 곤충병원성 곰팡이 메타리지움 아니소플리애 SD4-2 균주와 뷰베리아 바시아나 SD15 균주'가 개시되어 있고, 한국공개특허 제2021-0012341호에는 '진드기에 방제효과를 갖는 Metarhizium anisopoliea JEF-214, Metarhizium anisopoliea JEF-279 또는 Metarhizium anisopoliea JEF-290, 이를 포함하는 진드기 방제용 조성물 및 이를 이용한 진드기 방제방법'이 개시되어 있으나, 본 발명의 '뿌리응애 방제능을 가지는 메타리지움 펨피기 111 균주 및 이의 용도'에 대해서는 기재된 바가 없다.Meanwhile, Korean Patent Publication No. 2016-0140560 discloses 'entomopathogenic fungi Metarhizium anisopliae SD4-2 strain and Beauveria bassiana SD15 strain with pest control and antibacterial activity', and Korean Patent Publication No. No. 2021-0012341 discloses 'Metarhizium anisopoliea JEF-214, Metarhizium anisopoliea JEF-279 or Metarhizium anisopoliea JEF-290, which have a tick control effect, a composition for controlling ticks containing the same, and a method for controlling ticks using the same.' There is no description of the invention 'Metarygium pempigi 111 strain with root mite control ability and its use'.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명자는 뿌리응애(Rhizoglyphus robini Claparede)와 같은 흡즙성 해충에 높은 살충성을 보이면서 저항성 발생이 거의 없는 생물적 방제 방법인 곤충병원성 곰팡이를 이용한 방제 방법을 개발하고자, 국내에서 분리·확보된 342개의 곤충병원성 곰팡이 균주들을 이용하여 뿌리응애에 대해 높은 살비력을 가지는 곰팡이 균주들을 선발한 결과, 메타리지움 펨피기(Metarhizium pemphigi) 111 균주가 뿌리응애에 대해 높은 살비 활성을 가지며, UV-B 내성이 우수함을 확인함으로써, 본 발명을 완성하였다.The present invention was developed in response to the above-mentioned needs, and the present inventor proposes a control method using entomopathogenic fungi, which is a biological control method that shows high insecticidal properties against sucking pests such as root mites ( Rhizoglyphus robini Claparede) and has little resistance. In order to develop a method, fungal strains with high killing ability against root mites were selected using 342 entomopathogenic fungal strains isolated and secured in Korea. As a result, 111 strains of Metarhizium pemphigi were found to be effective against root mites. The present invention was completed by confirming that it has high acaricidal activity and excellent UV-B resistance.
상기 과제를 해결하기 위해, 본 발명은 응애 방제능 및 UV-B 내성을 가지는 메타리지움 펨피기(Metarhizium pemphigi) 111 균주(기탁번호: KACC 83052BP)를 제공한다.In order to solve the above problems, the present invention provides Metarhizium pemphigi 111 strain (Accession number: KACC 83052BP), which has mite control ability and UV-B resistance.
또한, 본 발명은 상기 균주 또는 이의 배양물을 유효성분으로 포함하는 응애 방제용 조성물을 제공한다.In addition, the present invention provides a composition for controlling mites containing the above strain or its culture as an active ingredient.
또한, 본 발명은 상기 조성물을 식물, 식물의 종자 또는 식물 식재 토양에 처리하는 단계를 포함하는, 응애 방제 방법을 제공한다.In addition, the present invention provides a method for controlling mites, comprising the step of treating the composition to plants, plant seeds, or plant planting soil.
본 발명의 메타리지움 펨피기 111 균주는 뿌리응애에 대해서만 특이적인 살충 효과를 보이므로, 인축이나 환경 생태계에 매우 안전한 해충 방제원으로서 사용될 수 있을 것이다.Since the Metarhizium Pempigi 111 strain of the present invention shows a specific insecticidal effect only against root mites, it can be used as a very safe pest control source for human livestock and the environmental ecosystem.
도 1은 곤충병원성 곰팡이 균주에 감염되어 죽은 뿌리응애 사충들을 보여주는 사진이다.
도 2는 곤충병원성 곰팡이 균주 11개의 뿌리응애(Rhizoglyphus robini Claparede)에 대한 7일간의 살비력을 나타내는 그래프이다. 대조구는 Tween-80 만을 처리한 것이고, 수직선은 표준오차를 나타낸다. 111, Metarhizium pemphigi 111; 1101, M. pemphigi 1101; 312, M. pingshaense 312; 441, M. pingshaense 441; 322, M. anisopliae 322; 432, M. anisopliae 432; 481, M. anisopliae 481; 4141, M. anisopliae 4141; 4183, M. anisopliae 4183; 423, M. anisopliae 423; 4312, M. anisopliae 4312.Figure 1 is a photograph showing dead root mite larvae infected with an entomopathogenic fungal strain.
Figure 2 is a graph showing the killing power for 7 days against root mites ( Rhizoglyphus robini Claparede) of 11 entomopathogenic fungal strains. The control group was treated with only Tween-80, and the vertical line represents the standard error. 111, Metarhizium pemphigi 111; 1101, M. pemphigi 1101; 312, M. pingshaense 312; 441, M. pingshaense 441; 322, M. anisopliae 322; 432, M. anisopliae 432; 481, M. anisopliae 481; 4141, M. anisopliae 4141; 4183, M. anisopliae 4183; 423, M. anisopliae 423; 4312, M. anisopliae 4312.
본 발명의 목적을 달성하기 위하여, 본 발명은 응애 방제능 및 UV-B 내성을 가지는 메타리지움 펨피기(Metarhizium pemphigi) 111 균주(기탁번호: KACC 83052BP)를 제공한다.In order to achieve the object of the present invention, the present invention provides Metarhizium pemphigi 111 strain (Accession number: KACC 83052BP), which has mite control ability and UV-B resistance.
본 발명의 메타리지움 펨피기 균주는 곤충병원성 곰팡이로, 곤충병원성 곰팡이는 곤충을 비롯한 무척추동물에 곰팡이병을 일으켜 숙주를 죽게 만들어 자연 상태에서 숙주의 밀도를 조절하는 역할을 하며, 곰팡이 종류에 따라서 병을 일으킬 수 있는 곤충이 제한적이기 때문에 목적으로 하는 해충에게만 피해를 주고 환경이나 다른 인축에 대한 독성은 없을 뿐만 아니라, 곤충에 의한 저항성 기작 역시 거의 발현되지 않는 것으로 보고되어 방제가 어려운 해충들의 방제를 위해 많은 연구 개발이 이루어지고 있다.The Metarhizium pemphigii strain of the present invention is an entomopathogenic fungus. Entomopathogenic fungi cause fungal disease in invertebrates, including insects, causing death of the host and play a role in controlling the density of the host in natural conditions, depending on the type of fungus. Since the number of insects that can cause disease is limited, it only causes damage to the target pests and is not toxic to the environment or other animals, and it is also reported that resistance mechanisms caused by insects are rarely expressed, making it possible to control pests that are difficult to control. A lot of research and development is being done for this purpose.
본 발명자들은 342개의 곤충병원성 곰팡이 균주들을 이용하여 뿌리응애(Rhizoglyphus robini Claparede)에 대해 높은 살비력을 가진 곰팡이를 분리한 결과, 메타리지움 속(Metarhizium spp.)에 속하는 11개 균주가 높은 살비력 및 우수한 UV-B 내성을 가지는 것을 확인하였으며, 이 중 메타리지움 펨피기 111 균주를 2021년 08월 20일자로 농업생명공학연구원(KACC)에 기탁하여 기탁 번호 KACC 83052BP를 부여받았다.The present inventors isolated fungi with high killing power against root mites ( Rhizoglyphus robini Claparede) using 342 entomopathogenic fungal strains. As a result, 11 strains belonging to the genus Metarhizium spp. had high killing power. And it was confirmed to have excellent UV-B resistance. Among these, Metarhizium Pempigi 111 strain was deposited with the Korea Agricultural and Biotechnology Research Institute (KACC) on August 20, 2021 and was given the deposit number KACC 83052BP.
본 발명의 메타리지움 펨피기 111 균주는 서열번호 1의 염기서열로 이루어진 ITS 영역 서열과, 서열번호 2의 염기서열로 이루어진 EF-1α 영역 서열의 동정을 통해 메타리지움 펨피기 균주로 확인되었다.The Metarhizium Pempigi 111 strain of the present invention was confirmed to be a Metarhizium Pempigi strain through identification of the ITS region sequence consisting of the nucleotide sequence of SEQ ID NO: 1 and the EF-1α region sequence consisting of the nucleotide sequence of SEQ ID NO: 2. .
본 발명은 또한, 상기 균주 또는 이의 배양물을 유효성분으로 포함하는 응애 방제용 조성물을 제공한다.The present invention also provides a composition for controlling mites containing the above strain or its culture as an active ingredient.
본 발명의 응애 방제용 조성물은 뿌리응애(Rhizoglyphus robini Claparede)에 높은 살비 활성을 가지는 메타리지움 펨피기(Metarhizium pemphigi) 111 균주(기탁번호: KACC 83052BP) 또는 이의 배양물을 유효성분으로 포함하여, 응애 방제 효과가 있다.The composition for controlling mites of the present invention contains Metarhizium pemphigi 111 strain (Accession number: KACC 83052BP) or a culture thereof, which has high acaricidal activity against root mites ( Rhizoglyphus robini Claparede), as an active ingredient, It is effective in controlling mites.
본 발명의 조성물에 있어서, 상기 '배양물'은 메타리지움 펨피기 111 균주의 배양물, 상기 배양물의 농축액, 또는 상기 배양물의 건조물을 포함할 수 있으나, 이에 제한되지 않는다.In the composition of the present invention, the 'culture' may include, but is not limited to, a culture of the Metarhizium pempigi 111 strain, a concentrate of the culture, or a dried product of the culture.
또한, 상기 배양물은 본 발명의 균주를 포함하는 배양물 자체의 형태로도 사용할 수 있으며, 또는 배양액 중의 배양배지를 제거하고 농축된 균체만을 회수하기 위해 원심분리 또는 여과과정을 거칠 수 있으며, 이러한 단계는 당업자가 필요에 따라 수행할 수 있다. 농축된 균체는 통상적인 방법에 따라 냉동(frozen)하거나 또는 냉동건조(lyophilized)하여 그 활성을 잃지 않도록 보존할 수 있다.In addition, the culture can be used in the form of the culture itself containing the strain of the present invention, or it can be subjected to centrifugation or filtration to remove the culture medium in the culture solution and recover only the concentrated cells. The steps can be performed as needed by those skilled in the art. Concentrated bacteria can be frozen or lyophilized according to conventional methods to preserve their activity.
상기 배양물은 통상적인 미생물의 배양방법에 의해 대량으로 배양하여 수득할 수 있으며, 배양배지로는 탄소원, 질소원, 비타민 및 미네랄을 포함하는 배지를 사용할 수 있으며, 상기 탄소원으로는 전분, 수크로즈, 시트레이트, 말토스, 글루코스, 만니톨, 글리세롤 또는 자일로스를 사용할 수 있으나, 이에 제한되지 않는다.The culture can be obtained by culturing in large quantities using a conventional microorganism culture method. A culture medium containing a carbon source, a nitrogen source, vitamins, and minerals can be used. The carbon source may include starch, sucrose, Citrate, maltose, glucose, mannitol, glycerol, or xylose can be used, but are not limited to these.
본 발명의 균주를 배양하는 방법은 당업계에 통상적으로 이용되는 방법에 따라 배양할 수 있으며, 특별한 방법에 한정되는 것은 아니다.The method of culturing the strain of the present invention can be cultured according to a method commonly used in the art, and is not limited to a special method.
본 발명의 균주를 배양하는 단계에서 얻어지는 상기 메타리지움 펨피기 111 균주 또는 이의 배양물을 첨가제로 사용할 경우, 상기 균주 또는 상기 균주의 배양물을 그대로 첨가하거나 다른 첨가제를 함께 사용할 수 있으며, 통상적인 방법에 따라 적절하게 사용될 수 있다. 성분의 혼합양은 그의 사용 목적에 따라 적합하게 결정될 수 있다.When using the Metarhizium Pempigi 111 strain or its culture obtained in the step of cultivating the strain of the present invention as an additive, the strain or the culture of the strain can be added as is or other additives can be used together, and the usual It can be used appropriately depending on the method. The mixing amount of ingredients can be appropriately determined depending on the purpose of use.
본 발명에 따른 응애 방제용 조성물은 예를 들어 직접 분사가능한 용액, 분말 및 현탁액의 형태 또는 고농축 수성, 유성 또는 다른 현탁액, 분산액, 에멀젼, 유성 분산액, 페이스트, 분진, 흩뿌림 물질 또는 과립제로 제조할 수 있으나, 이에 제한되지는 않는다. 또한, 상기 식물병 방제용 조성물은 분사, 분무, 살포, 흩뿌림 또는 붓기에 의해 사용될 수 있다. 사용 형태는 의도한 목적에 의존하는데, 모든 경우에 본 발명에 따른 조성물의 분포가 가능한 한 미세하고 균일하도록 해야 한다.The compositions for controlling mites according to the invention can be prepared, for example, in the form of directly sprayable solutions, powders and suspensions or as highly concentrated aqueous, oily or other suspensions, dispersions, emulsions, oily dispersions, pastes, dusts, scattering substances or granules. However, it is not limited to this. Additionally, the composition for controlling plant diseases can be used by spraying, spraying, spraying, scattering or pouring. The form of use depends on the intended purpose; in all cases it should be ensured that the distribution of the composition according to the invention is as fine and uniform as possible.
또한, 본 발명의 응애 방제용 조성물은 다양한 형태로 제제화할 수 있다. 상기 제제는 예를 들어 용매 및/또는 담체를 첨가함으로써 제조될 수 있다. 종종, 비활성 첨가제 및 표면-활성 물질, 예를 들어 유화제 또는 분산제를 제제에 혼합한다. 적합한 표면-활성 물질은 방향족 술폰산(예를 들어 리그노술폰산, 페놀-술폰산, 나프탈렌- 및 디부틸나프탈렌술폰산), 지방산, 알킬- 및 알킬아릴술포네이트, 알킬 라우릴 에테르, 지방 알코올 술페이트의 알칼리 금속, 알카라인 토금속, 암모늄염, 술페이트화 헥사-, 헵타- 및 옥타데칸올, 지방 알코올 글리콜에테르의 염, 술포네이트 나프탈렌 및 이의 유도체, 포름알데히드의 축합물, 나프탈렌 또는 나프탈렌술폰산, 페놀 및 포름알데히드의 축합물, 폴리옥시에틸렌옥틸 페놀 에테르, 에톡실화 이소옥틸-, 옥틸- 또는 노닐페놀, 알킬페닐 또는 트리부틸페닐 폴리글리콜 에테르, 알킬아릴폴리에테르 알코올, 이소트리데실 알코올, 지방 알코올/에틸렌 옥사이드 축합물, 에톡실화 피마자유, 폴리옥시에틸렌 알킬에테르 또는 폴리옥시프로필렌, 라우릴 알코올 폴리글리콜 에테르 아세테이트, 소르비톨 에스테르, 리그닌-술파이트 폐액 또는 메틸셀룰로오스일 수 있으나, 이에 제한되지 않는다.Additionally, the composition for controlling mites of the present invention can be formulated in various forms. The formulation may be prepared, for example, by adding solvents and/or carriers. Often, inert additives and surface-active substances, such as emulsifiers or dispersants, are mixed into the formulation. Suitable surface-active substances include aromatic sulfonic acids (e.g. lignosulfonic acid, phenol-sulfonic acid, naphthalene- and dibutylnaphthalenesulfonic acid), fatty acids, alkyl- and alkylarylsulfonates, alkyl lauryl ethers, alkalis of fatty alcohol sulfates. Metals, alkaline earth metals, ammonium salts, sulfated hexa-, hepta- and octadecanols, salts of fatty alcohol glycol ethers, sulfonate naphthalene and its derivatives, condensates of formaldehyde, naphthalene or naphthalenesulfonic acid, phenol and formaldehyde. Condensates, polyoxyethyleneoctyl phenol ethers, ethoxylated isooctyl-, octyl- or nonylphenol, alkylphenyl or tributylphenyl polyglycol ethers, alkylarylpolyether alcohols, isotridecyl alcohol, fatty alcohol/ethylene oxide condensates. , ethoxylated castor oil, polyoxyethylene alkyl ether or polyoxypropylene, lauryl alcohol polyglycol ether acetate, sorbitol ester, lignin-sulfite waste liquid, or methylcellulose, but is not limited thereto.
적합한 고형 담체 물질은 원칙적으로, 모두 다공성이고, 농업적으로 허용가능한 담체, 예를 들어 광물토류(예컨대 실리카, 실리카 겔, 실리케이트, 활석, 고령토, 석회암, 석회, 초크, 보울, 황토, 점토류, 백운석, 규조 토류, 황산칼슘, 황산 마그네슘, 산화마그네슘, 분쇄 합성물질), 비료(예컨대 황산암모늄, 인산암모늄, 질산암모늄, 우레아), 식물성 제품(예컨대 곡물 가루, 나무 껍질 가루, 목분(wood meal) 및 견과 껍질 가루) 또는 셀룰로오스 분말일 수 있으나, 이에 제한되지는 않는다. 또한, 상기 고형 담체는 1종류 또는 2종류 이상을 혼합하여 사용할 수도 있다.Suitable solid carrier materials are, in principle, all porous and agriculturally acceptable carriers, for example mineral earths (e.g. silica, silica gel, silicates, talc, kaolin, limestone, lime, chalk, bowl, red clay, clay, Dolomite, diatomaceous earth, calcium sulfate, magnesium sulfate, magnesium oxide, ground synthetics), fertilizers (e.g. ammonium sulfate, ammonium phosphate, ammonium nitrate, urea), vegetable products (e.g. grain flour, bark meal, wood meal) and nut shell powder) or cellulose powder, but is not limited thereto. Additionally, the above solid carrier may be used alone or in a mixture of two or more types.
본 발명의 응애 방제용 조성물은 유효성분으로서 메타리지움 펨피기 111 균주를 단독으로, 또는 2종 이상의 다른 살비제(acaricide) 물질 등과 혼합하여 사용할 수 있다.The composition for controlling mites of the present invention can be used as an active ingredient, Metarhizium pempigi 111 strain alone or in combination with two or more other acaricide substances.
또한, 상기 응애 방제용 조성물의 제조 방법은 당업계에 공지된 임의의 방법을 이용할 수 있으며, 특정 방법에 특별히 제한되는 것은 아니다.Additionally, the method for producing the composition for controlling mites may use any method known in the art, and is not particularly limited to a specific method.
본 발명의 응애 방제용 조성물은 식물체 흡수 및 효과를 증진시키기 위하여 확산제 및 침투제, 또는 계면활성제와도 혼용이 가능하다.The composition for controlling mites of the present invention can be mixed with dispersants, penetrants, or surfactants to enhance plant absorption and effectiveness.
본 발명은 또한, 상기 조성물을 식물, 식물의 종자 또는 식물 식재 토양에 처리하는 단계를 포함하는, 응애 방제 방법을 제공한다.The present invention also provides a method for controlling mites, comprising the step of applying the composition to plants, plant seeds, or plant planting soil.
본 발명에 따른 응애 방제 방법에 있어서, 상기 조성물은 뿌리응애(Rhizoglyphus robini Claparede)에 높은 살비 활성을 가지는 메타리지움 펨피기(Metarhizium pemphigi) 111 균주(기탁번호: KACC 83052BP) 또는 이의 배양물을 유효성분으로 포함한다.In the mite control method according to the present invention, the composition is effective against Metarhizium pemphigi 111 strain (Accession number: KACC 83052BP) or a culture thereof, which has high acaricidal activity against root mites ( Rhizoglyphus robini Claparede). Included as an ingredient.
본 발명의 상기 응애 방제 방법은, 상기 메타리지움 펨피기 111 균주 또는 이의 배양물을 유효성분으로 포함하는 조성물의 유효량을 식물에 침지하거나 관주, 즉, 분무하여 수행할 수 있다. 침지하는 방법의 경우, 상기 조성물의 유효량을 식물체 주변의 토양에 붓거나 또는 종자를 상기 조성물의 유효량에 담가둘 수 있다.The mite control method of the present invention can be carried out by immersing or drenching, that is, spraying, an effective amount of a composition containing the Metarhizium pempigi 111 strain or its culture as an active ingredient on the plant. In the case of the soaking method, an effective amount of the composition can be poured into the soil around the plant or seeds can be soaked in an effective amount of the composition.
본 발명의 '유효량'은 유익한 또는 원하는 결과를 일으키기에 충분한 양으로, 응애, 특히 뿌리응애(R. robini Claparede)를 방제하기 위해 상기 메타리지움 펨피기 111 균주 또는 이의 배양물을 물로 균일하게 희석한 후 동력살포기와 같은 적절한 살포장치를 이용하여 식물체 및 경작지에 살포할 수 있다.The 'effective amount' of the present invention is an amount sufficient to produce a beneficial or desired result, which is obtained by uniformly diluting the Metarhizium pempigee 111 strain or its culture with water to control mites, especially root mites ( R. robini Claparede). After that, it can be sprayed on plants and farmland using an appropriate spray device such as a power sprayer.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by examples. However, the following examples only illustrate the present invention, and the content of the present invention is not limited to the following examples.
재료 및 방법Materials and Methods
점박이응애spotted mite
뿌리응애(R. robini)는 양파를 공급하고 온도 25℃, 상대습도 80-90%, 광조건 12L:12D 조건으로 실험실 조건하에서 사육하면서 실험에 이용하였다.Root mites ( R. robini ) were used in experiments by supplying onions and rearing them under laboratory conditions at a temperature of 25°C, relative humidity of 80-90%, and light conditions of 12L:12D.
곤충병원성 곰팡이 균주Entomopathogenic fungal strains
1) 곰팡이 균주의 배양1) Cultivation of fungal strains
우리나라 전국 토양에서 분리된 342개의 곰팡이 균주(신 등, Biocontrol Science and Technology 2013, 23(3):288-304)를 사용하였다(표 1).342 fungal strains (Shin et al., Biocontrol Science and Technology 2013, 23(3):288-304) isolated from soil across Korea were used (Table 1).
곰팡이 균주는 감자한천배지(potato dextrose agar, PDA) 플레이트상에서 25℃에서 14일 동안 배양한 다음, PDA 플레이트에서 14일된 곰팡이 분생포자를 긁어내고 0.05% Tween-80 (Difco, USA) 용액에 물질을 재현탁시켜 수집하였다. 균사 잔해물을 제거하기 위해 상기 분생포자 현탁액을 격렬하게 교반하고 면 거즈를 통해 여과시켰다. 여과된 포자는 혈구계산기를 사용하여 농도를 측정하였다.The fungal strain was cultured on potato dextrose agar (PDA) plates at 25°C for 14 days, then 14-day-old fungal conidia were scraped from the PDA plates and the material was placed in 0.05% Tween-80 (Difco, USA) solution. It was resuspended and collected. The conidial suspension was stirred vigorously and filtered through cotton gauze to remove mycelial debris. The concentration of filtered spores was measured using a hemocytometer.
2) 곰팡이 균주의 동정2) Identification of fungal strains
뿌리응애에 병원성이 확인된 균주들의 동정은 현미경적 관찰을 통한 포자의 모양 및 색을 통한 형태학적 동정과 함께 분자생물학적 동정을 실시하였다. 분자생물학적 동정을 위해서 곰팡이의 게놈 DNA 추출은 PDA에서 2주간 자란 곰팡이 균체 일부를 fungal DNA extraction buffer (0.2 M Tris-Cl (pH 7.5), 0.5 M Nacl, 10 nM EDTA (pH 8.0) and 1%(w/v) SDS)를 처리한 후 phenol-chloroform-isoamyl alcohol (25:24:1)로 원심분리하여 DNA를 정제하였다. 정제된 DNA 용액은 냉에탄올을 이용하여 침전시키고 원심분리 후 멸균 증류수에 녹여 실험에 이용하였다.Identification of strains confirmed to be pathogenic to root mites was performed through morphological identification through the shape and color of spores through microscopic observation, as well as molecular biological identification. For molecular biological identification, the genomic DNA of fungi was extracted by extracting some fungal cells grown on PDA for 2 weeks in fungal DNA extraction buffer (0.2 M Tris-Cl (pH 7.5), 0.5 M Nacl, 10 nM EDTA (pH 8.0) and 1% ( w/v) SDS) and then centrifuged with phenol-chloroform-isoamyl alcohol (25:24:1) to purify DNA. The purified DNA solution was precipitated using cold ethanol, centrifuged, dissolved in sterile distilled water, and used in experiments.
분리 균주의 분자생물학적 동정을 위한 PCR 프라이머는 internal transcribed spacer (ITS1-5.8S-ITS2) 부분의 증폭을 위한 ITS1(5'-TCCGTAGGTGAACCTGCGG-3', 서열번호 3)과 ITS4 (5'-TCCTCCGCTTATTGATATGC-3', 서열번호 4) 프라이머를 사용하였으며, Elongation factor-1 aplha 부분의 증폭을 위해서는 1577F(5'-CARGAYGTBTACAAGATYGGTGG-3', 서열번호 5)와 2218R(5'-ATGACACCRACRGCRACRGTYTG-3', 서열번호 6) 프라이머를 사용하였다. PCR 반응은 AccuPower PCR PreMix (Bioneer Co., Korea)를 이용하여 변성(denaturation) 94℃ 5분, 결합(annealing) 55℃ 30초, 신장(extension) 72℃ 1분의 35회 반복 조건으로 Thermal Cycler (TaKaRa, Japan)를 이용하여 수행하였다.PCR primers for molecular biological identification of the isolated strain are ITS1 (5'-TCCGTAGGTGAACCTGCGG-3', SEQ ID NO: 3) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3) for amplification of the internal transcribed spacer (ITS1-5.8S-ITS2). ', SEQ ID NO: 4) primers were used, and for amplification of the Elongation factor-1 aplha portion, 1577F (5'-CARGAYGTBTACAAGATYGGTGG-3', SEQ ID NO: 5) and 2218R (5'-ATGACACCRACRGCRACRGTYTG-3', SEQ ID NO: 6) Primers were used. The PCR reaction was performed using AccuPower PCR PreMix (Bioneer Co., Korea) using a thermal cycler under the following conditions: denaturation at 94°C for 5 minutes, annealing at 55°C for 30 seconds, and extension at 72°C for 1 minute. This was performed using (TaKaRa, Japan).
PCR 반응 후 증폭 산물은 1.0% 아가로스 겔을 이용하여 전기영동 분석하고 Power Gel Extraction Kit (Dyne Bio Inc., Korea)를 이용하여 순수 정제하였다. 정제된 PCR 산물은 Macrogen (Korea)사에 direct sequencing 의뢰하여 염기서열을 분석하였다. 염기서열은 Multalin (http://multalin.toulouse.inra.fr/multalin/)를 이용하여 정렬 한 다음 BLAST search tool을 이용하여 기 보고된 다른 곰팡이들 서열과 비교 분석하였다.After the PCR reaction, the amplification product was analyzed by electrophoresis using a 1.0% agarose gel and purified using the Power Gel Extraction Kit (Dyne Bio Inc., Korea). The purified PCR product was subjected to direct sequencing by Macrogen (Korea) and its base sequence was analyzed. The base sequences were aligned using Multalin (http://multalin.toulouse.inra.fr/multalin/) and then compared and analyzed with other previously reported fungal sequences using the BLAST search tool.
생물학적 검정(Bioassay)Bioassay
1) 뿌리응애 병원성 곰팡이 분리1) Isolation of pathogenic fungi from root mites
뿌리응애에 병원성을 가진 곰팡이를 분리하기 위하여 배양 14일 된 곰팡이 포자를 생물 검정에 사용하였다. 생존율이 90%를 초과하는 포자 현탁액을 생물 검정에 사용하였다. 포자의 생존력은 SDAY+B 배지(Saboraud dextrose agar, 1% Yeast extract, 0.05% benomyl, pH 5.6)에서 결정되었다.To isolate fungi pathogenic to root mites, 14-day-old fungal spores were used in a bioassay. Spore suspensions with viability exceeding 90% were used for bioassays. Spore viability was determined on SDAY+B medium (Saboraud dextrose agar, 1% yeast extract, 0.05% benomyl, pH 5.6).
342개의 곰팡이 균주들을 무작위로 크게 4개 그룹으로 나누고, 각 그룹의 곰팡이 균주들의 포자 현탁액을 하나로 합쳐서 생물검정에 사용하였다. 생물검정은 각 그룹의 곰팡이 포자현탁액을 뿌리응애 성충 50마리에 침지법으로 접종하고, 접종된 뿌리응애는 10일 동안 매일 관찰하면서 각각의 사충을 수집하였다. 뿌리응애 사충들은 표피에서 곰팡이의 발생을 육안으로 확인된 경우에만 곰팡이에 의한 치사로 인정하였다.The 342 fungal strains were randomly divided into four groups, and the spore suspensions of the fungal strains from each group were combined into one and used for bioassay. For the bioassay, the fungal spore suspension of each group was inoculated into 50 adult root mites by immersion, and the inoculated root mites were observed daily for 10 days and each dead mite was collected. Root mite death was recognized as being caused by mold only when the growth of mold on the epidermis was visually confirmed.
사멸한 뿌리응애 사충으로부터 병원성 곰팡이를 분리하기 위해 사충에서 곰팡이 포자를 멸균된 이쑤시개로 수거하여 0.02% Tween-80 용액에 현탁한 후, 곤충병원성 곰팡이 선택배지인 SDA-D50 (sabouraud dextrose agar, 100 ㎍/㎖ chloramphenicol, 50 ㎍/㎖ streptomycin, 50 ㎍/㎖ dodine)에 도말하였다. 이 후 25℃, 암 조건에서 7일간 배양하면서 형성된 콜로니를 분리하여, 다시 PDA배지에 접종하여 곰팡이를 순수 분리하고 형태학적 및 분자생물학적 동정을 완료 한 후 다음 실험에 이용하였다.To isolate pathogenic fungi from dead root mite larvae, fungal spores were collected from the larvae with a sterilized toothpick, suspended in 0.02% Tween-80 solution, and then cultured on SDA-D50 (sabouraud dextrose agar, 100 ㎍, a selective medium for entomopathogenic fungi). /㎖ chloramphenicol, 50㎍/㎖ streptomycin, 50㎍/㎖ dodine). Afterwards, colonies formed while culturing for 7 days at 25°C in dark conditions were isolated and inoculated onto PDA medium to pure isolate the fungi. After completing morphological and molecular biological identification, the colonies were used in the next experiment.
2) 곰팡이의 살비 활성2) Acaricidal activity of fungi
뿌리응애에 대해 병원성이 있는 것으로 확인된 곰팡이 균주들은 정량적 병원력 검정을 위하여 PDA (potato dextrose agar)배지에서 2주 동안 배양하여 포자 생성을 유도한 후, 생성된 포자를 0.05% Tween-80을 이용하여 포자현탁액을 만들고 혈구계산기를 이용하여 계수하고 실험에 사용하였다. 생물검정 전 포자의 생존률은 모두 90%이상인 것을 사용하였다. 각 곰팡이 균주들의 포자 농도는 1 x 107 포자/㎖로 하여 30마리의 뿌리응애에 침지 접종하였다. 접종 후 7일 동안 매일 사충을 관찰하며 수거하였고, 사충의 표면에서 곰팡이 발생이 육안으로 확인된 경우에만 곰팡이에 의한 치사인 것으로 인정하였다. 대조구로는 0.05% Tween-80 용액만을 뿌리응애에 처리하였다.Fungal strains confirmed to be pathogenic against root mites were cultured on PDA (potato dextrose agar) medium for 2 weeks to induce spore production for quantitative pathogenicity testing, and then the generated spores were cultured using 0.05% Tween-80. A spore suspension was made, counted using a hemocytometer, and used in the experiment. All spores with a survival rate of more than 90% before bioassay were used. The spore concentration of each fungal strain was 1 x 10 7 spores/ml, and 30 root mites were inoculated by immersion. After inoculation, dead worms were observed and collected every day for 7 days, and death due to mold was recognized only when mold growth was visually confirmed on the surface of the dead worm. As a control, only 0.05% Tween-80 solution was applied to root mites.
곰팡이 포자의 UV-B 내성 분석Analysis of UV-B resistance of fungal spores
곰팡이 균주들의 포자의 UV-B 내성을 평가하기 위하여 포자 현탁액 20 ㎕ (5 x 106 conidia/㎖)를 spreading 하지 않고 SDA-B 배지에 플레이팅하였다. 포자(conidia)를 25℃, UV 조사 챔버(Bio-Link-BLX-E254, France) 내에서 0.1 또는 0.2 J cm-2의 방사 조도에 노출시키고, 25℃에서 배양하였다. 대조구는 UV-B는 조사하지 않고, 동일 배지에서 동일 조건으로 배양하였다. 발아율은 무처리 대조구 대비 처리구의 발아 수준을 비교하여 계산하였다. 최소 100개의 포자가 각 실험의 매 회차에 사용되었으며, 삼반복 수행하였다.To evaluate the UV-B resistance of spores of fungal strains, 20 μl (5 x 10 6 conidia/ml) of spore suspension was plated on SDA-B medium without spreading. Spores (conidia) were exposed to irradiance of 0.1 or 0.2 J cm -2 in a UV irradiation chamber (Bio-Link-BLX-E254, France) at 25°C and cultured at 25°C. The control group was cultured under the same conditions in the same medium without UV-B irradiation. Germination rate was calculated by comparing the germination level of the treated group with that of the untreated control group. A minimum of 100 spores were used in each round of each experiment, and three replicates were performed.
통계분석Statistical analysis
살비활성과 열안정성 평가는 3회 반복 수행하였으며, 결과 값은 SPSS 통계프로그램으로 유의수준 0.05 이하로 유의성 검정을 하였다. 자료는 평균±표준오차(SE)로 표시하였다.The evaluation of acaricidal activity and thermal stability was repeated three times, and the results were tested for significance using the SPSS statistical program at a significance level of 0.05 or less. Data were expressed as mean ± standard error (SE).
실시예 1. 점박이응애 병원성 곰팡이 분리Example 1. Isolation of spotted mite pathogenic fungi
1) 병원성 곰팡이 분리1) Isolation of pathogenic mold
곤충병원성 곰팡이 342개 균주를 4개 그룹으로 묶고 각 그룹에 대하여 포자 현탁액을 만들어 뿌리응애에 대해 병원성을 검정하였다. 그 결과, 전체 200마리의 뿌리응애 중에서 38마리의 응애에서 곰팡이에 의한 치사를 확인하였다.342 strains of entomopathogenic fungi were grouped into four groups, and spore suspensions were prepared for each group to test their pathogenicity against root mites. As a result, out of a total of 200 root mites, 38 were confirmed to be killed by the fungus.
곰팡이에 의한 치사가 확인된 뿌리응애 사충으로부터 곰팡이를 분리한 결과, 57개의 균주를 분리하였다. 분리된 균주들에 대해서는 포자의 모양과 색깔에 의한 형태학적 동정과 함께 ITS 영역과 Elongation factor-1 aplha 영역에 대한 분자생물학적 동정을 실시하였다. 그 결과, 뷰베리아 바시아나(Beauveria bassiana) 29개 균주, 메타리지움 아니소플리애(Metarhizium anisopliae) 19개 균주, 메타리지움 펨피기(M. pemphigi) 5개 균주, 메타리지움 핑샤엔스(M. pingshaense) 2개 균주, 아칸토마이세스 아테누아투스(Akanthomyces attenuatus) 1개 균주와 아칸토마이세스 베시컬러(A. versicolor) 1개 균주가 동정되었다(표 2 내지 표 3 참조).As a result of isolating fungi from root mite larvae in which death by fungi was confirmed, 57 strains were isolated. The isolated strains were subjected to morphological identification based on the shape and color of the spores, as well as molecular biological identification of the ITS region and Elongation factor-1 aplha region. As a result, 29 strains of Beauveria bassiana , 19 strains of Metarhizium anisopliae, 5 strains of Metarhizium pemphigi ( M. Two strains of M. pingshaense , one strain of Akanthomyces attenuatus and one strain of A. versicolor were identified (see Tables 2 to 3).
동정된 균주 57개를 이용하여 뿌리응애(Rhizoglyphus robini)에 대해서 병원성을 재검정한 결과, 15개 균주에서만 병원성이 다시 확인되었으며, 그 중 분자생물학적 동정 결과가 100% 일치하는 균주들은 동일한 균주로 판단하여 한 개의 균주만 다음 실험에 이용하였다. 최종적으로 분리한 균주 중 메타리지움 아니소플리애(M. anisopliae) 7개 균주, 메타리지움 펨피기(M. pemphigi) 2개 균주, 메타리지움 핑샤엔스(M. pingshaense) 2개 균주를 선발하였다.As a result of re-testing the pathogenicity of Rhizoglyphus robini using 57 identified strains, pathogenicity was confirmed again in only 15 strains, and among them, strains with 100% matching molecular biological identification results were judged to be the same strain. Therefore, only one strain was used in the next experiment. Among the strains finally isolated, 7 strains of Metarhizium anisopliae ( M. anisopliae ), 2 strains of Metarhizium pemphigi ( M. pemphigi ), and 2 strains of Metarhizium pingshaense ( M. pingshaense ) selected.
실시예 2. 점박이응애에 대한 살비력Example 2. Killing power against spotted mites
뿌리응애에 대해 병원성이 확인된 11개 균주들간의 살비력을 비교 평가하였다(도 1). 그 결과, 11개 균주의 뿌리응애에 대한 살비력은 균주에 따라 다양하게 나타났으며, 처리 후 7일에 38%에서부터 100%까지 나타났다. 그 중에서 메타리지움 아니소플리애 432와 4312 균주, 그리고 메타리지움 펨피기 111 균주가 100%의 살비력을 보였다. 이 중 메타리지움 아니소플리애 432 및 메타리지움 펨피기 111 균주는 처리 후 5일에도 99% 이상의 높은 살비력을 보였다.The killing power of 11 strains confirmed to be pathogenic against root mites was compared and evaluated (Figure 1). As a result, the killing power against root mites of 11 strains varied depending on the strain, ranging from 38% to 100% at 7 days after treatment. Among them, Metarhizium anisopliae strains 432 and 4312, and Metarhizium pempgii 111 strains showed 100% killing power. Among these, Metarhizium anisopliae 432 and Metarhizium pempigi 111 strains showed high killing power of more than 99% even 5 days after treatment.
실시예 3. 곰팡이 포자의 UV-B 내성Example 3. UV-B resistance of fungal spores
뿌리응애에 대해 7일차에 100% 살비력이 확인된 3개의 균주(메타리지움 아니소플리애 432와 4312, 메타리지움 펨피기 111)의 포자에 대해 UV-B 내성을 비교 평가하였다. 그 결과, 하기 표 4와 같이 UV-B 0.1 J cm-2 처리 조건에서 메타리지움 펨피기 111 균주가 9.0±2.0%의 포자 발아율을 보여 메타리지움 아니소플리애 균주들에 비해 UV-B 내성이 높은 것을 알 수 있었다.UV-B resistance was compared and evaluated on the spores of three strains (Metharrhizium anisopliae 432 and 4312, Metarhizium pempgii 111), which were confirmed to have 100% killing ability against root mites on day 7. As a result, as shown in Table 4 below, Metarhizium Pempigi 111 strain showed a spore germination rate of 9.0 ± 2.0% under UV-B 0.1 J cm -2 treatment conditions, showing UV-B compared to Metarhizium anisopliae strains. It was found that tolerance was high.
이상의 결과들을 종합하여, 메타리지움 펨피기(M. pemphigi) 111 균주가 뿌리응애에 대해서 높은 살비력과 UV-B 내성을 가지고 있어 뿌리응애 방제에 유용한 균주임을 확인하였다. 메타리지움 펨피기 111 균주는 농업생명공학연구원(Korean Agricultural Culture Collection, KACC)에 2021년 08월 20일자로 기탁을 진행하였으며, 기탁기관으로부터 기탁번호 KACC 83052BP를 부여받았다.Combining the above results, it was confirmed that Metarhizium pemphigi ( M. pemphigi ) 111 strain has high acaricidal power and UV-B resistance against root mites and is a useful strain for controlling root mites. Metarhizium Pempigi 111 strain was deposited with the Korean Agricultural Culture Collection (KACC) on August 20, 2021, and was assigned the deposit number KACC 83052BP by the depositing institution.
<110> Chungbuk National University Industry-Academic Cooperation Foundation <120> Metarhizium pemphigi 111 strain for controlling bulb mite and uses thereof <130> PN21383 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 541 <212> DNA <213> Unknown <220> <223> Metarhizium pemphigi <400> 1 agggatcatt accgagttta caactcccaa acccaatgtg aactatacct gtctaccgtt 60 gcctcggcgg gttcgcccgc cgagggaccg acaaataaac tcttgtattt ctatctttag 120 catgtctgag tggaatcata aacaaatgaa tcaaaacttt caacaacgga tctcttggtt 180 ctggcatcga tgaagaacgc agcgaaatgc gataagtaat gtgaattgca gaattcagtg 240 aatcatcgaa tctttgaacg cacattgcgc ccgccagtat tctggcgggc atgcctgttc 300 gagcgtcatt acaaccctca agccccccgg cttggtgttg gggaccggcc accggtgccc 360 tgctgcttcc gcggcaggcg cacccggccg cccccgaaat gaattggcgg ccccgtcgcg 420 gcctccctct gcgtagtagc acacatctcg cagctggagc gcggcgcggc cactgccgta 480 aaacgcacca acttttttta cagttgacct cgaatcaggt aggaataccc gctgaactta 540 a 541 <210> 2 <211> 517 <212> DNA <213> Unknown <220> <223> Metarhizium pemphigi <400> 2 tattggaact gtccctgtcg gccgtatcga gactggtgtc ctcaagcccg gtatggtcgt 60 taccttcgct ccctccaacg tcaccactga agtcaagtcc gtggagatgc accacgagca 120 gctttctgag ggtgtccccg gtgacaacgt tggtttcaac gtgaagaacg tttctgtcaa 180 ggaaatccgc cgtggtaacg ttgctggtga ctccaagaac gaccccccca tgggcgccgc 240 ttctttcgat gcccaggtca tcgttctcaa ccaccccggc caggtcggtg ctggttacgc 300 tcccgtcctc gattgtcaca ccgcccacat tgcctgcaag ttctccgaga tcaaggagaa 360 gattgaccga cgtaccggta aggctgttga gtctgccccc aagttcatca agtccggtga 420 ctctgccatc gtcaagatgg ttccctccaa gcccatgtgc gttgaggctt tcaccgatta 480 ccctcccctg ggccgtttcg ccgtccgtga tatgcgt 517 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 tccgtaggtg aacctgcgg 19 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 tcctccgctt attgatatgc 20 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 cargaygtbt acaagatygg tgg 23 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 atgacaccra crgcracrgt ytg 23 <110> Chungbuk National University Industry-Academic Cooperation Foundation <120> Metarhizium pemphigi 111 strain for controlling bulb mite and uses it <130> PN21383 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 541 <212> DNA <213> Unknown <220> <223> Metarhizium pemphigi <400> 1 agggatcatt accgagttta caactcccaa acccaatgtg aactatacct gtctaccgtt 60 gcctcggcgg gttcgcccgc cgagggaccg acaaataaac tcttgtattt ctatctttag 120 catgtctgag tggaatcata aacaaatgaa tcaaaacttt caacaacgga tctcttggtt 180 ctggcatcga tgaagaacgc agcgaaatgc gataagtaat gtgaattgca gaattcagtg 240 aatcatcgaa tctttgaacg cacattgcgc ccgccagtat tctggcgggc atgcctgttc 300 gagcgtcatt acaaccctca agccccccgg cttggtgttg gggaccggcc accggtgccc 360 tgctgcttcc gcggcaggcg cacccggccg cccccgaaat gaattggcgg ccccgtcgcg 420 gcctccctct gcgtagtagc acacatctcg cagctggagc gcggcgcggc cactgccgta 480 aaacgcacca acttttttta cagttgacct cgaatcaggt aggaataccc gctgaactta 540 a 541 <210> 2 <211> 517 <212> DNA <213> Unknown <220> <223> Metarhizium pemphigi <400> 2 tatggaact gtccctgtcg gccgtatcga gactggtgtc ctcaagcccg gtatggtcgt 60 taccttcgct ccctccaacg tcaccactga agtcaagtcc gtggagatgc accacgagca 120 gctttctgag ggtgtccccg gtgacaacgt tggtttcaac gtgaagaacg tttctgtcaa 180 ggaaatccgc cgtggtaacg ttgctggtga ctccaagaac gaccccccca tgggcgccgc 240 ttctttcgat gcccaggtca tcgttctcaa ccaccccggc caggtcggtg ctggttacgc 300 tcccgtcctc gattgtcaca ccgcccacat tgcctgcaag ttctccgaga tcaaggagaa 360 gattgaccga cgtaccggta aggctgttga gtctgccccc aagttcatca agtccggtga 420 ctctgccatc gtcaagatgg ttccctccaa gcccatgtgc gttgaggctt tcaccgatta 480 ccctcccctg ggccgtttcg ccgtccgtga tatgcgt 517 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 tccgtaggtg aacctgcgg 19 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 tcctccgctt attgatatgc 20 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 cargaygtbt acaagatygg tgg 23 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 atgacaccra crgcracrgt ytg 23
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