KR20240020308A - Novel Burkholderia sp. having nematicidal activity and use thereof - Google Patents
Novel Burkholderia sp. having nematicidal activity and use thereof Download PDFInfo
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- KR20240020308A KR20240020308A KR1020220096930A KR20220096930A KR20240020308A KR 20240020308 A KR20240020308 A KR 20240020308A KR 1020220096930 A KR1020220096930 A KR 1020220096930A KR 20220096930 A KR20220096930 A KR 20220096930A KR 20240020308 A KR20240020308 A KR 20240020308A
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- nematodes
- culture
- burkholderia
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Abstract
발명은 살선충 활성을 갖는 토양 유래 신규한 버크홀데리아 속 균주; 상기 균주, 이의 파쇄물, 이의 추출물, 이의 배양물, 상기 배양물의 농축액, 상기 배양물의 건조물로 이루어진 군에서 선택된 하나 이상을 포함하는 기생 선충 방제용 조성물 또는 식물 생장 촉진용 미생물 제제; 기생 선충 방제 방법에 관한 것으로, 살선충 미생물 방제제에 대한 개발 및 생산에 있어 유용하게 활용되어, 친환경 농업의 구현뿐만 아니라, 농작물의 농약 잔류 및 토양 오염을 경감시켜 환경 보전에 기여할 수 있다.The invention relates to a novel soil-derived strain of the genus Burkholderia with nematicidal activity; A composition for controlling parasitic nematodes or a microbial preparation for promoting plant growth comprising at least one selected from the group consisting of the strain, its lysate, its extract, its culture, a concentrate of the culture, and a dried product of the culture; It relates to a method for controlling parasitic nematodes, and can be usefully used in the development and production of nematode microbial control agents, contributing to environmental conservation by not only implementing eco-friendly agriculture but also reducing pesticide residues and soil contamination in crops.
Description
본 발명은 살선충 활성을 갖는 토양 유래 신규한 버크홀데리아 속 균주; 상기 균주, 이의 파쇄물, 이의 추출물, 이의 배양물, 상기 배양물의 농축액, 상기 배양물의 건조물로 이루어진 군에서 선택된 하나 이상을 포함하는 기생 선충 방제용 조성물 또는 식물 생장 촉진용 조성물; 미생물 제제; 기생 선충 방제 방법에 관한 것이다.The present invention relates to a novel Burkholderia genus strain derived from soil having nematicidal activity; A composition for controlling parasitic nematodes or a composition for promoting plant growth comprising at least one selected from the group consisting of the strain, its lysate, its extract, its culture, a concentrate of the culture, and a dried product of the culture; microbial preparations; It relates to a method for controlling parasitic nematodes.
선충(Nematode)은 동식물, 바닷물, 민물, 토양 속에서 생활하면서 동물이나 식물에 기생하기도 한다. 식물에 기생하는 선충은 수백여 종에 이르며 주로 토양, 잎, 줄기, 열매를 통해 침입한다. 침입한 기생 선충은 식물의 양분을 탈취하여 직접피해를 주거나 곰팡이, 세균 등 다른 병원체의 침입을 조장하여 간접피해를 일으킨다.Nematodes live in animals and plants, seawater, freshwater and soil, and sometimes become parasites on animals and plants. There are hundreds of species of nematodes that live on plants, and they mainly invade through the soil, leaves, stems, and fruits. Invading parasitic nematodes cause direct damage by stealing nutrients from plants, or cause indirect damage by encouraging the invasion of other pathogens such as molds and bacteria.
대표적인 선충인 뿌리혹선충 (Meloidogyne sp.)은 광범위한 작물에 심각한 피해를 입히는 식물 기생성 선충이다. 특히, 고구마 뿌리혹선충 (Meloidogyne incognita), 땅콩 뿌리혹선충 (M. arenaria), 당근 뿌리혹선충 (M. hapla) 및 자바니카 뿌리혹선충(M. javanica)은 전세계 농업지대에 널리 분포하며 경제적으로 심각한 피해를 끼치는 고 있는 중요한 해충이며 550여종의 식물이 기주로 기록되어있다. 뿌리혹선충은 2령 유충 시기에 식물의 뿌리 생장점 근처로 침입하여 식물의 뿌리 내에서 기생하면서 성장하고, 이 과정에서 선충이 분비하는 호르몬의 작용으로 식물의 뿌리가 혹 모양으로 변형된다. 이에 따른 양분의 불균형으로 식물의 영양실조 및 뿌리의 양분과 수분 흡수기능이 저하되어 식물의 성장 및 생육을 직간접적으로 방해하게 되며 경제적 손실을 일으킨다. 이러한 식물 기생 선충으로 인한 피해규모는 세계 농업생산력 년 12% 감소로 이어지며, 피해액은 약 1,300억 달러로 추정된다. 또한, 뿌리혹선충은 식물에 직접적인 피해를 줄 뿐만 아니라, 다른 식물 기생 선충 및 다른 병원균과 상호 작용하여 질병 복합체를 형성하여 추가적인 피해를 일으킨다. Root-knot nematode (Meloidogyne sp.), a representative nematode, is a plant parasitic nematode that causes serious damage to a wide range of crops. In particular, sweet potato root-knot nematodes (Meloidogyne incognita), peanut root-knot nematodes (M. arenaria), carrot root-knot nematodes (M. hapla), and Javanica root-knot nematodes (M. javanica) are widely distributed in agricultural areas around the world and cause serious economic damage. It is an important pest and over 550 species of plants are recorded as hosts. Root-knot nematodes invade near the root growth point of a plant during the second instar larval stage and grow parasitic within the plant's roots. During this process, the roots of the plant are transformed into a nodule shape by the action of hormones secreted by the nematode. The resulting nutrient imbalance leads to malnutrition of the plant and a decrease in the nutrient and water absorption function of the roots, which directly or indirectly hinders the growth and development of the plant and causes economic loss. The scale of damage caused by these plant parasitic nematodes leads to a 12% annual decrease in global agricultural productivity, and the damage is estimated at approximately $130 billion. In addition, root-knot nematodes not only cause direct damage to plants, but also interact with other plant parasitic nematodes and other pathogens to form disease complexes, causing additional damage.
이러한 선충을 방제하기 위한 대표적인 예로 메틸브로마이드와 같은 여러 훈증제와 선충탄(Fosthiazate)와 같은 여러 높은 독성의 화학 살선충제가 주로 사용되어 왔다. 하지만 화학 살선충제의 독성에 따른 생태계 영향 및 환경 잔류성 등의 부담으로 인하여 뿌리혹선충의 예방 및 방제를 위한 새로운 환경친화적인 생물학적 방제제의 개발이 점점 더 중요해지고 있다. 이에 따라 최근 환경 친화적인 생물학적 방제제를 개발하려는 연구가 전 세계적으로 활발하게 진행되고 있으며, 기술적 돌파 필요성이 요구되고 있는 실정이다. 최근 몇 년 동안, 해외 각국에서는 생물학적 방제제에 대한 연구가 지속되고 있으나 (일본 공개특허 제 2015-551526호), 국내의 경우, 현재 다수의 제품이 화학 살선충제로 이루어져 있으며 효율적으로 선충을 방제할 수 있는 미생물 제제는 거의 없는 실정이다.Representative examples for controlling these nematodes include fumigants such as methyl bromide and highly toxic chemical nematicides such as fosthiazate. However, due to the burden of ecological effects and environmental persistence due to the toxicity of chemical nematicides, the development of new environmentally friendly biological control agents for the prevention and control of root-knot nematodes is becoming increasingly important. Accordingly, research to develop environmentally friendly biological control agents has recently been actively conducted around the world, and the need for technological breakthroughs is required. In recent years, research on biological control agents has continued in overseas countries (Japanese Patent Publication No. 2015-551526), but in Korea, many products currently consist of chemical nematicides and are not effective in controlling nematodes. There are very few microbial preparations that can be used.
이러한 배경 하에, 본 발명자들은 살선충 활성이 높고 작물 생육 촉진능이 우수한 생물학적 방제제에 관한 연구를 수행하였으며, 토양으로부터 분리한 신규한 버크홀데리아 속 균주는 살선충 효과가 있을 뿐만 아니라, 작물 생육 촉진 효과가 현저히 우수함을 확인함으로써 본 발명을 완성하였다. Against this background, the present inventors conducted research on biological control agents with high nematicidal activity and excellent crop growth promotion ability, and the novel Burkholderia genus strain isolated from soil not only has a nematicidal effect but also promotes crop growth. The present invention was completed by confirming that the effect was significantly excellent.
본 발명의 하나의 목적은 기탁번호 KCTC14975BP로 기탁된 버크홀데리아 JB-1 (Burkholderia JB-1) 균주 또는 기탁번호 KCTC14976BP로 기탁된 버크홀데리아 JB-2 (Burkholderia JB-2) 균주를 제공하는 것이다. One object of the present invention is to provide a Burkholderia JB-1 strain deposited with accession number KCTC14975BP or a Burkholderia JB-2 strain deposited with accession number KCTC14976BP. .
본 발명의 다른 하나의 목적은 상기 균주, 상기 균주의 파쇄물, 상기 균주의 추출물, 상기 균주의 배양물, 상기 배양물의 농축액 및 상기 배양물의 건조물로 이루어진 군에서 선택된 하나 이상을 포함하는, 기생 선충 방제용 조성물을 제공하는 것이다. Another object of the present invention is to control parasitic nematodes, comprising at least one selected from the group consisting of the strain, lysate of the strain, extract of the strain, culture of the strain, concentrate of the culture, and dried material of the culture. To provide a composition for use.
본 발명의 또 다른 하나의 목적은 상기 기생 선충 방제용 조성물을 식물체 또는 기생 선충에 처리하는 단계를 포함하는, 기생 선충 방제 방법을 제공하는 것이다. Another object of the present invention is to provide a method for controlling parasitic nematodes, comprising treating plants or parasitic nematodes with the composition for controlling parasitic nematodes.
본 발명의 또 다른 하나의 목적은 상기 균주, 상기 균주의 파쇄물, 상기 균주의 추출물, 상기 균주의 배양물, 상기 배양물의 농축액 및 상기 배양물의 건조물로 이루어진 군에서 선택된 하나 이상을 유효성분으로 포함하는, 식물 생장 촉진용 조성물을 제공하는 것이다.Another object of the present invention is to contain as an active ingredient at least one selected from the group consisting of the strain, the lysate of the strain, the extract of the strain, the culture of the strain, the concentrate of the culture, and the dried material of the culture. , to provide a composition for promoting plant growth.
본 발명의 또 다른 하나의 목적은 상기 기생 선충 방제용 또는 식물 생장 촉진용 조성물을 포함하는 미생물제제를 제공하는 것이다. Another object of the present invention is to provide a microbial agent containing the composition for controlling parasitic nematodes or promoting plant growth.
본 발명의 또 다른 하나의 목적은 상기 식물 생장 촉진용 조성물을 식물의 종자, 잎, 줄기, 뿌리 또는 이에 근접한 토양에 직접 살포하거나 침지시키는 것을 포함하는, 식물 생장 촉진하는 방법을 공하는 것이다. Another object of the present invention is to provide a method for promoting plant growth, which includes spraying or immersing the composition for plant growth promotion directly on the seeds, leaves, stems, roots of plants, or soil adjacent thereto.
이를 구체적으로 설명하면 다음과 같다. 한편, 본 발명에서 개시된 각각의 설명 및 실시형태는 각각의 다른 설명 및 실시 형태에도 적용될 수 있다. 즉, 본 발명에서 개시된 다양한 요소들의 모든 조합이 본 발명의 범주에 속한다. 또한, 하기 기술된 구체적인 서술에 위하여 본 발명의 범주가 제한된다고 볼 수 없다. This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Additionally, the scope of the present invention cannot be considered limited to the specific description described below.
본 발명의 하나의 양태는 기탁번호 KCTC14975BP로 기탁된 버크홀데리아 JB-1 (Burkholderia JB-1) 균주 또는 기탁번호 KCTC14976BP로 기탁된 버크홀데리아 JB-2 (Burkholderia JB-2) 균주를 제공한다. One aspect of the present invention provides a Burkholderia JB-1 strain deposited with accession number KCTC14975BP or a Burkholderia JB-2 strain deposited with accession number KCTC14976BP.
상기 균주는 살선충 활성, 생장 촉징 활성, 키틴 및 단백질 분해 활성을 갖는다. The strain has nematicidal activity, growth promoting activity, chitin and proteolytic activity.
본 발명의 용어, "버크홀데리아 (Burkholderia)"는 버크홀데리아과 (Burkholderiaceae)의 그람 음성, 산소성의 세균 속에 속한다. 버크홀데리아 종은 발견되는 서식지가 토양부터 사람의 호흡기까지 매우 다양하며, 일부 종은 잠재적 병원성을 지니고 있다. 본 발명의 버크홀데리아는 밭 토양 또는 산림 토양에서 유래한 것일 수 있으나, 이에 제한되지 않는다. As used herein, the term “Burkholderia” belongs to the genus of Gram-negative, oxygenated bacteria of the Burkholderiaceae family. Burkholderia species are found in a wide variety of habitats, from soil to the human respiratory tract, and some species are potentially pathogenic. Burkholderia of the present invention may be derived from field soil or forest soil, but is not limited thereto.
본 발명의 "살선충 활성"은 선충을 죽이는 효과를 가지고 있는 활성을 의미하며, 식물체가 생장하는 토양에서 선충의 밀도의 감소와 상기 선충의 알 부화를 억제시키는 효과뿐만 아니라, 피해 식물체에 발생하는 혹과 난낭의 개수가 감소하는 효과를 의미한다. "Nematicidal activity" of the present invention refers to an activity that has the effect of killing nematodes, and not only has the effect of reducing the density of nematodes in the soil in which plants grow and suppressing the hatching of eggs of the nematodes, but also the effect of suppressing the hatching of nematode eggs in the soil in which plants grow. This refers to the effect of reducing the number of lumps and egg sacs.
본 발명에서 선충은 고구마 뿌리혹선충(Meloidogyne incognita), 땅콩 뿌리혹선충 (M. arenaria), 당근 뿌리혹선충 (M. hapla), 자바니카 뿌리혹선충(M. javanica), 잎선충류(Aphelenchodies), 줄기선충류(Ditylenchus), 씨스트선충(Globodera, Heterodera), 뿌리썩이선충류(Pratylenchus), 궁침선충류(Trichodorus), 검선충류(Xiphinema) 또는 상기 선충의 알 일 수 있으나, 이에 제한되지 않는다.In the present invention, nematodes include sweet potato root-knot nematodes (Meloidogyne incognita), peanut root-knot nematodes (M. arenaria), carrot root-knot nematodes (M. hapla), Javanica root-knot nematodes (M. javanica), leaf nematodes (Aphelenchodies), and stem nematodes ( Ditylenchus, cyst nematodes (Globodera, Heterodera), Pratylenchus, Trichodorus, Xiphinema, or eggs of the above nematodes, but are not limited thereto.
본 발명의 균주는 살선충 활성뿐만 아니라, 토양 및 식물체와 연관된 해충인 곰팡이, 박테리아, 곤충, 거미류, 선충, 민달팽이, 달팽이 등 및 이들 해충의 알 또는 유충, 농작물, 자연환경의 토양 해충 및 이들의 알 및 유충에 대한 살충, 살연체동물, 살균 또는 살박테리아 등의 활성을 가질 수 있다. 구체적으로, 이집트숲모기(Aedes aeypti), 흰줄숲모기(Aedes albopictus), 빨간집모기(Culex pipiens), 작은뿌리파리(Bradysia agrestis), 파밤나방(Spodoptera exigua), 배추좀나방(Plutella xylostella), 담배거세 미나방(Spodoptera litura), 미국흰불나방(Hyphantria cunea), 왕담배나방(Helicoverpa armigera), 화랑곡나방 (Plodia interpunctella), 이화명나방(Chilo suppressalis), 혹명나방(Cnaphalocrocis medinalis), 옥수수조명나방(Ostrinia furnacalis), 톱다리개미허리노린재(Riptortus clavatus), 벼물바구미(Lissorhoptrus oryzophilus), 벼멸구(Nilaparvata lugens), 애멸구(Laodelphax striatellus), 목화진딧물(Aphis gossypii), 복숭아혹진딧물(Myzus persicae), 오이총채벌레(Thrips palmi), 꽃노랑총채벌레(Frankliniella occidentalis), 담배가루이(Bemisia tabaci) 등 유충호르몬이 존재하는 절지동물 또는 상기 해충의 알 또는 유충에 대한 살충, 살연체동물, 살균 또는 살박테리아 활성일 수 있으나, 이에 제한되지 않는다. 상기와 같은 살선충, 살충, 살연체동물, 살균, 살박테리아 등의 활성을 바탕으로 본 발명의 JB-1 또는 JB-2 균주는 식물병 방제 효과를 가질 수 있으나, 이에 제한되지 않는다. The strain of the present invention not only has nematicidal activity, but also protects against fungi, bacteria, insects, arachnids, nematodes, slugs, snails, etc., which are pests associated with soil and plants, and eggs or larvae of these pests, agricultural crops, soil pests in the natural environment, and their It may have insecticidal, molluscan, bactericidal, or bactericidal activities against eggs and larvae. Specifically, Aedes aeypti, Aedes albopictus, Culex pipiens, Bradysia agrestis, Spodoptera exigua, Plutella xylostella, Spodoptera litura, Hyphantria cunea, Helicoverpa armigera, Plodia interpunctella, Chilo suppressalis, Cnaphalocrocis medinalis, corn moth ( Ostrinia furnacalis), Riptortus clavatus, Lissorhoptrus oryzophilus, Nilaparvata lugens, Laodelphax striatellus, Aphis gossypii, Myzus persicae, cucumber shoots Insecticidal, molluscan, bactericidal, or bactericidal activity against arthropods containing larval hormones, such as Thrips palmi, thrips (Frankliniella occidentalis), and whiteflies (Bemisia tabaci), or eggs or larvae of these pests. It may be, but is not limited to this. Based on the nematicidal, insecticidal, mollusk-killing, bactericidal, and bactericidal activities described above, the JB-1 or JB-2 strain of the present invention may have a plant disease control effect, but is not limited thereto.
본 발명의 살선충 활성을 확인할 수 있는 식물은 식물의 전체, 뿌리, 줄기, 잎, 및 종자와 같은 식물의 일부, 식물체의 세포 또는 조직일 수 있다. 그 예로, 곡물(밀, 보리, 호밀, 귀리, 쌀, 사탕 수수 등), 비트(사탕무 및 사료 사탕무), 과일류 (사과, 배, 자두, 복숭아, 아몬드, 체리, 딸기, 라스베리, 블랙 베리, 수박), 콩과 식물(콩, 렌즈 콩, 완두콩, 콩), 오일 식물(유채, 겨자, 양귀비, 올리브, 해바라기, 코코넛, 피마자 오일 플랜트, 코코아 콩, 땅콩 등), 오이 식물(오이, 참외, 멜론 등), 섬유 식물(면, 아마, 대마, 황마), 감귤류의 과일(귤, 오렌지, 레몬, 자몽 등), 야채(시금치, 상추, 아스파라거스, 양배추, 양파, 토마토, 감자, 파프리카, 호박 등), 녹나무과(아보카도, 당근, 계피, 녹나무), 활엽수와 침엽수(린든 - 트리, 주목 나무, 떡갈 나무, 오리 나무, 포플러, 자작 나무, 전나무, 낙엽송, 소나무), 옥수수, 담배, 견과류, 커피, 사탕 수수, 차, 덩굴, 홉, 바나나, 천연 고무와 같은 식물, 또는 관상용 식물의 전체 또는 일부일 수 있으나, 선충에 의해 뿌리 혹이 야기되거나 변형 또는 식물의 성장 및 생육을 방해 받은 식물이라면 특별히 제한되지 않는다. Plants that can confirm the nematicidal activity of the present invention may be whole plants, parts of plants such as roots, stems, leaves, and seeds, and cells or tissues of plants. Examples include grains (wheat, barley, rye, oats, rice, sugar cane, etc.), beets (sugar and fodder beets), fruits (apples, pears, plums, peaches, almonds, cherries, strawberries, raspberries, blackberries, etc.) watermelon), legumes (beans, lentils, peas, soybeans), oil plants (rapeseed, mustard, poppy, olive, sunflower, coconut, castor oil plant, cocoa bean, peanut, etc.), cucumber plants (cucumber, melon, Melon, etc.), fiber plants (cotton, flax, hemp, jute), citrus fruits (tangerines, oranges, lemons, grapefruit, etc.), vegetables (spinach, lettuce, asparagus, cabbage, onions, tomatoes, potatoes, paprika, pumpkin, etc.) ), Lauraceae (avocado, carrot, cinnamon, camphor), broad-leaved and coniferous trees (linden-tree, yew, oak, alder, poplar, birch, fir, larch, pine), corn, tobacco, nuts, coffee, It may be the whole or part of plants such as sugarcane, tea, vines, hops, bananas, natural rubber, or ornamental plants, but is not particularly limited as long as it is a plant that has root nodules caused by nematodes or has been deformed or whose growth and growth has been disturbed by nematodes. No.
본 발명의 균주는 살선충 관련 효소 활성은 단백질분해효소, 키틴분해효소, 젤라틴분해효소, 콜라겐분해효소 활성일 수 있으나, 이에 제한되지 않는다. The nematode-related enzyme activity of the strain of the present invention may be protease, chitinase, gelatinase, or collagenase, but is not limited thereto.
본 발명의 일 구현예에서는, 신규한 버크홀데리아 속 JB-1 또는 JB-2 균주를 처리하여 선충의 표피 및 알의 주된 성분인 단백질 및 키틴에 대한 분해 여부를 비교하였으며, 그 결과 버크홀데리아 속 JB-1 및 JB-2 균주의 단백질 및 키틴에 대한 기질 특이적 효소 분해능을 관찰함으로써 JB-1 또는 JB-2 균주의 살선충 활성이 우수함을 확인하였다. In one embodiment of the present invention, the novel Burkholderia genus JB-1 or JB-2 strains were treated to compare the decomposition of proteins and chitin, which are the main components of the epidermis and eggs of nematodes, and as a result, Burkholderia By observing the substrate-specific enzymatic decomposition ability of proteins and chitin of the JB-1 and JB-2 strains, it was confirmed that the JB-1 or JB-2 strains had excellent nematicidal activity.
본 발명의 용어, "식물 생장"은 식물의 씨앗을 발아하여 뿌리·줄기·잎을 내거나 또는 점차 크기와 무게를 더해가는 과정을 의미하므로, "식물 생장 촉진"은 식물의 씨앗의 발아 또는 발아된 식물의 크기와 무게의 증가를 촉진하는 것을 의미한다. 본 발명의 균주는 살선충 활성뿐만 아니라, 식물의 생장을 촉진하는 활성을 보유하며, 효과적인 미생물 제제로 이용될 수 있다. As the term of the present invention, "plant growth" refers to the process of germinating plant seeds to produce roots, stems, and leaves, or gradually increasing size and weight, "promoting plant growth" refers to the process of germinating plant seeds or sprouting them. It is meant to promote an increase in the size and weight of plants. The strain of the present invention possesses not only nematicidal activity but also activity that promotes plant growth, and can be used as an effective microbial agent.
본 발명의 다른 하나의 양태는 상기 균주, 상기 균주의 파쇄물, 상기 균주의 추출물, 상기 균주의 배양물, 상기 배양물의 농축액 및 상기 배양물의 건조물로 이루어진 군에서 선택된 하나 이상을 포함하는, 기생 선충 방제용 조성물을 제공한다. Another aspect of the present invention is to control parasitic nematodes, comprising at least one selected from the group consisting of the strain, lysate of the strain, extract of the strain, culture of the strain, concentrate of the culture, and dried material of the culture. Provides a composition for
본 발명의 용어, "균주(strain)"은 한 개의 세포에서 유래한, 무성생식을 통해 증식하여 동질의 유전적 특징을 갖는 개체들의 집합을 의미하며, 하나의 종(species) 안에는 유전적 특징이 서로 다른 여러 개의 균주들(genetic variants)이 존재한다. 본 발명에서 균주는 버크홀데리아 속을 의미하며, 구체적으로 기탁번호 KCTC14975BP로 기탁된 버크홀데리아 JB-1 (Burkholderia JB-1) 또는 기탁번호 KCTC14976BP로 기탁된 버크홀데리아 JB-2 (Burkholderia JB-2)일 수 있으나, 이에 제한되지 않는다. 구체적으로, 상기 균주는 서열번호 3 또는 4의 16s rRNA 서열을 포함하는 균주일 수 있으나, 이에 제한되지 않는다. As used herein, the term “strain” refers to a collection of individuals derived from a single cell, proliferated through asexual reproduction, and having identical genetic characteristics. Within a species, genetic characteristics are not present. Several different genetic variants exist. In the present invention, strain refers to the genus Burkholderia, specifically, Burkholderia JB-1 deposited with accession number KCTC14975BP or Burkholderia JB-2 deposited with accession number KCTC14976BP. 2), but is not limited to this. Specifically, the strain may be a strain containing the 16s rRNA sequence of SEQ ID NO: 3 or 4, but is not limited thereto.
본 발명에서, 상기 균주는 버크홀데리아 속 균주의 균체, 건조된 균체, 파쇄물 등을 포함할 수 있다. 이 때, 상기 건조된 균체는 분무 건조균, 동결 건조균, 진공 건조균, 드럼 건조균 등의 건조균일 수 있고, 상기 파쇄물은 균주 자체를 화학적 또는 물리적 힘에 의하여 균주의 세포벽을 파쇄하여 얻은 산물일 수 있다.In the present invention, the strain may include bacterial cells, dried bacterial cells, shredded material, etc. of strains of the genus Burkholderia. At this time, the dried bacteria may be dried bacteria such as spray-dried bacteria, freeze-dried bacteria, vacuum-dried bacteria, drum-dried bacteria, etc., and the shredded material is a product obtained by crushing the cell wall of the strain by chemical or physical force. It can be.
본 발명의 용어, "추출물"은 본 발명에서 제공하는 균주를 추출 처리하여 얻어지는 추출액, 상기 추출액의 희석액이나 농축액, 상기 추출액을 건조하여 얻어지는 건조물, 상기 추출액의 조정제물이나 정제물, 또는 이들의 혼합물 등, 추출액 자체 및 추출액을 이용하여 형성 가능한 모든 제형의 추출물을 포함한다. 상기 추출물을 얻는 추출 방법은 특별히 제한되지 않으며, 당해 기술 분야에서 통상적으로 사용하는 방법에 따라 추출할 수 있다. 상기 추출 방법의 비제한적인 예로는, 열수 추출법, 초음파 추출법, 여과법, 환류 추출법 등을 들 수 있으며, 이들은 단독으로 수행되거나 2종 이상의 방법을 병용하여 수행될 수 있다. 상기 추출물을 얻는 데 사용되는 추출 용매의 종류는 특별히 제한되지 않으며, 당해 기술 분야에서 공지된 임의의 용매를 사용할 수 있다. 상기 추출 용매의 비제한적인 예로는 물, 알코올, 에틸아세테이트 또는 이들의 혼합 용매 등을 들 수 있으며, 이들은 단독으로 사용되거나 1종 이상 혼합하여 사용될 수 있다.The term "extract" in the present invention refers to an extract obtained by extracting and processing the strain provided in the present invention, a diluted or concentrated liquid of the extract, a dried product obtained by drying the extract, a crude product or purified product of the extract, or a mixture thereof. etc., includes the extract itself and all formulations of the extract that can be formed using the extract. The extraction method for obtaining the extract is not particularly limited, and may be extracted according to a method commonly used in the art. Non-limiting examples of the extraction method include hot water extraction, ultrasonic extraction, filtration, and reflux extraction, which may be performed alone or by combining two or more methods. The type of extraction solvent used to obtain the extract is not particularly limited, and any solvent known in the art can be used. Non-limiting examples of the extraction solvent include water, alcohol, ethyl acetate, or a mixed solvent thereof, and these may be used alone or in combination of one or more types.
본 발명의 일 구현예에서는, 본 발명의 버크홀데리아 속 JB-1, JB-2 균주와 버크홀데리아 속에 속하는 다른 22종 균주 및 살충제로 알려진 선충탄 (fosthiazate)의 뿌리혹선충에 대한 활성을 검정한 결과, 버크홀데리아 속 JB-1, JB-2는 다른 버크홀데리아 속 균주 대비 현저히 우수한 살선충 활성을 가지며, 양성대조군인 선충탄과 활성이 유사하거나 이보다 우수한 살선충 활성을 가지는 것을 확인하였다. In one embodiment of the present invention, the activity of the Burkholderia genus JB-1 and JB-2 strains of the present invention, the other 22 strains belonging to the Burkholderia genus, and fosthiazate, a known insecticide, against root-knot nematodes is tested. As a result, it was confirmed that Burkholderia genus JB-1 and JB-2 have significantly superior nematicidal activity compared to other Burkholderia genus strains, and that they have similar or better nematicidal activity than the positive control nematode charcoal. .
본 발명의 용어, "배양물"은 상기 균주를 배지에서 배양하여 수득한 산물을 의미한다. 그 예로 본 발명의 배양물은 버크홀데리아 속의 배양 배지에서 상기 균주를 수확한 후 남은 배지에 잔존한 성분을 포함할 수 있다.As used herein, the term “culture” refers to a product obtained by culturing the above strain in a medium. For example, the culture of the present invention may contain components remaining in the culture medium remaining after harvesting the strain from the culture medium of the Burkholderia genus.
상기 배양물은 상기 버크홀데리아 속 균주의 전체 배양물, 배양상등액, 파쇄물, 이들의 분획물 등이 될 수 있다. 이때, 상기 배양상등액은 상기 균주의 배양물을 원심분리하여 수득할 수 있고, 상기 파쇄물은 상기 균주를 물리적으로 또는 초음파처리하여 수득할 수 있으며, 상기 분획물은 상기 배양물, 배양상등액, 파쇄물 등을 원심분리, 크로마토그래피 등의 방법에 적용하여 수득할 수 있다.The culture may be the entire culture of the Burkholderia genus strain, culture supernatant, lysate, fractions thereof, etc. At this time, the culture supernatant can be obtained by centrifuging the culture of the strain, the lysate can be obtained by physically or sonicating the strain, and the fraction is the culture, culture supernatant, lysate, etc. It can be obtained by applying methods such as centrifugation and chromatography.
본 발명의 균주의 배양에 사용되는 배지 및 기타 배양 조건은 통상의 버크홀데리아 속 미생물의 배양에 사용되는 배지라면 특별한 제한 없이 어느 것이나 사용될 수 있으며, 구체적으로는 본 발명의 균주를 적당한 탄소원, 질소원, 인원, 무기화합물, 아미노산 및/또는 비타민 등을 함유한 통상의 배지 내에서 호기성 또는 혐기성 조건 하에서 온도, pH 등을 조절하면서 배양할 수 있다.The medium and other culture conditions used for cultivating the strain of the present invention can be any medium used for cultivating common microorganisms of the Burkholderia genus without particular limitation, and specifically, the strain of the present invention can be used as an appropriate carbon source and nitrogen source. , can be cultured under aerobic or anaerobic conditions in a conventional medium containing phosphorus, inorganic compounds, amino acids and/or vitamins, etc., while controlling temperature, pH, etc.
본 발명에서 상기 탄소원으로는 글루코오스, 프룩토오스, 수크로오스, 말토오스 등과 같은 탄수화물; 만니톨, 소르비톨 등과 같은 당 알코올, 피루브산, 락트산, 시트르산 등과 같은 유기산; 글루타메이트, 메티오닌, 라이신 등과 같은 아미노산 등이 포함될 수 있으나, 이에 제한되지 않는다. 또한, 전분 가수분해물, 당밀, 블랙스트랩 당밀, 쌀겨울, 카사버, 사탕수수 찌꺼기 및 옥수수 침지액 같은 천연의 유기 영양원을 사용할 수 있으며, 글루코오스 및 살균된 전처리 당밀(즉, 환원당으로 전환된 당밀) 등과 같은 탄수화물이 사용될 수 있고, 그 외의 적정량의 탄소원을 제한 없이 다양하게 이용할 수 있다. 이들 탄소원은 단독으로 사용되거나 2 종 이상이 조합되어 사용될 수 있다.In the present invention, the carbon source includes carbohydrates such as glucose, fructose, sucrose, maltose, etc.; Sugar alcohols such as mannitol, sorbitol, etc., organic acids such as pyruvic acid, lactic acid, citric acid, etc.; Amino acids such as glutamate, methionine, lysine, etc. may be included, but are not limited thereto. Additionally, natural organic nutrient sources such as starch hydrolyzate, molasses, blackstrap molasses, rice bran, cassava, bagasse and corn steep liquor can be used, as well as glucose and pre-sterilized molasses (i.e. molasses converted to reducing sugars). Carbohydrates such as the like can be used, and various other carbon sources in appropriate amounts can be used without limitation. These carbon sources may be used individually or two or more types may be used in combination.
상기 질소원으로는 암모니아, 황산암모늄, 염화암모늄, 초산암모늄, 인산암모늄, 탄산안모늄, 질산암모늄 등과 같은 무기질소원; 아미노산, 펩톤, NZ-아민, 육류 추출물, 효모 추출물, 맥아 추출물, 옥수수 침지액, 카세인 가수분해물, 어류 또는 그의 분해생성물, 탈지 대두 케이크 또는 그의 분해 생성물 등과 같은 유기 질소원이 사용될 수 있다. 이들 질소원은 단독으로 사용되거나 2 종 이상이 조합되어 사용될 수 있으나, 이에 제한되지 않는다.The nitrogen source includes inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, anmonium carbonate, and ammonium nitrate; Organic nitrogen sources such as amino acids, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its decomposition products, defatted soybean cake or its decomposition products, etc. can be used. These nitrogen sources may be used alone or in combination of two or more types, but are not limited thereto.
상기 인원으로는 인산 제1칼륨, 인산 제2칼륨, 또는 이에 대응되는 소디움-함유 염 등이 포함될 수 있다. 무기화합물로는 염화나트륨, 염화칼슘, 염화철, 황산마그네슘, 황산철, 황산망간, 탄산칼슘 등이 사용될 수 있다.The agent may include monopotassium phosphate, dipotassium phosphate, or a corresponding sodium-containing salt. Inorganic compounds include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate.
그 외에 상기 배지에는 아미노산, 비타민 및/또는 적절한 전구체 등이 포함될 수 있다. 구체적으로, 상기 균주의 배양 배지에는 L-아미노산 등이 첨가될 수 있다. 구체적으로는 글리신(glycine), 글루타메이트(glutamate), 및/또는 시스테인(cysteine) 등이 첨가될 수 있고, 필요에 따라서는 라이신(lysine) 등의 L-아미노산 이 더 첨가될 수 있으나 반드시 이에 제한되지 않는다.In addition, the medium may contain amino acids, vitamins, and/or appropriate precursors. Specifically, L-amino acids, etc. may be added to the culture medium of the strain. Specifically, glycine, glutamate, and/or cysteine may be added, and if necessary, L-amino acids such as lysine may be added, but are not necessarily limited thereto. No.
상기 배지 또는 전구체는 배양물에 회분식 또는 연속식으로 첨가될 수 있으며, 이에 제한되지 않는다.The medium or precursor may be added to the culture in a batch or continuous manner, but is not limited thereto.
본 발명에서, 균주의 배양 중에 수산화암모늄, 수산화칼륨, 암모니아, 인산, 황산 등과 같은 화합물을 배양물에 적절한 방식으로 첨가하여, 배양물의 pH를 조정할 수 있다. 또한, 배양 중에는 지방산 폴리글리콜 에스테르와 같은 소포제를 사용하여 기포 생성을 억제할 수 있다. 또한, 배양물의 호기 상태를 유지하기 위하여, 배양물 내로 산소 또는 산소 함유 기체를 주입하거나 혐기 및 미호기 상태를 유지하기 위해 기체의 주입 없이 혹은 질소, 수소 또는 이산화탄소 가스를 주입할 수 있다.In the present invention, the pH of the culture can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. to the culture in an appropriate manner during cultivation of the strain. Additionally, during culturing, foam generation can be suppressed by using an antifoaming agent such as fatty acid polyglycol ester. Additionally, to maintain the aerobic state of the culture, oxygen or oxygen-containing gas can be injected into the culture, or to maintain the anaerobic and microaerobic state, nitrogen, hydrogen, or carbon dioxide gas can be injected without the injection of gas.
본 발명의 일 구현예에서는, 버크홀데리아 속 JB-1 또는 JB-2 균주 배양물을 식물에 처리한 후 식물 성장 및 난낭 수를 조사한 결과, 무처리 및 선충탄을 처리한 식물 대비 식물의 지상부 길이, 지상부 및 뿌리 무게가 현저히 증가함을 확인하였고, 뿌리 무게 당 난낭 수 또한 적게 나타남으로써, 버크홀데리아 속 JB-1 또는 JB-2 균주는 식물 생육 촉진 효과뿐만 아니라, 살선충 효과가 우수함을 확인하였다. In one embodiment of the present invention, after treating plants with a culture of strains of the Burkholderia genus JB-1 or JB-2, plant growth and number of egg sacs were examined, as a result of examining the above-ground parts of the plants compared to untreated and nematode-treated plants. It was confirmed that the length, aerial part, and root weight were significantly increased, and the number of egg cysts per root weight was also small, showing that Burkholderia genus JB-1 or JB-2 strains not only have a plant growth promoting effect, but also have an excellent nematicidal effect. Confirmed.
또한, 버크홀데리아 속 JB-1 또는 JB-2 균주 배양물을 처리한 경우, 식물 방어 관련 유전자의 발현량이 현저히 증가하였고, 그 결과 살리실산, 자스몬산 관련된 방어 유전자 및 활성산소 관련 유전자가 활성화되었음을 확인하였다. In addition, when cultures of Burkholderia genus JB-1 or JB-2 strains were treated, the expression level of plant defense-related genes was significantly increased, and as a result, it was confirmed that salicylic acid, jasmonic acid-related defense genes and reactive oxygen species-related genes were activated. did.
본 발명의 용어, "기생 선충 방제"는 기생하는 선충의 생장을 억제하거나, 죽이는 일로서, 살선충 활성을 의미한다. The term "parasitic nematode control" in the present invention refers to the activity of inhibiting the growth or killing of parasitic nematodes and means nematode activity.
상기 "살선충 활성"은 전술한 바와 같다. The “nematicidal activity” is as described above.
본 발명의 다른 하나의 양태는 상기 기생 선충 방제용 조성물을 식물체 또는 기생 선충에 처리하는 단계를 포함하는 기생 선충 방제 방법을 제공한다. Another aspect of the present invention provides a method for controlling parasitic nematodes, comprising treating plants or parasitic nematodes with the composition for controlling parasitic nematodes.
상기 “버크홀데리아", "기생 선충 방제" 및 "살선충 활성"은 전술한 바와 같다. “Burkholderia”, “parasitic nematode control”, and “nematicidal activity” are the same as described above.
본 발명의 다른 하나의 양태는 균주, 상기 균주의 파쇄물, 상기 균주의 추출물, 상기 균주의 배양물, 상기 배양물의 농축액 및 상기 배양물의 건조물로 이루어진 군에서 선택된 하나 이상을 유효성분으로 포함하는, 식물 생장 촉진용 조성물을 제공한다. Another aspect of the present invention is a plant comprising as an active ingredient one or more selected from the group consisting of a strain, a lysate of the strain, an extract of the strain, a culture of the strain, a concentrate of the culture, and a dried product of the culture. A composition for promoting growth is provided.
상기 "버크홀데리아", "균주", "파쇄물", "추출물", "배양물" 및 "식물 생장 촉진"은 전술한 바와 같다.The terms “Burkholderia”, “strain”, “disaggregate”, “extract”, “culture” and “plant growth promotion” are as described above.
본 발명의 기생 선충 방제 방법 또는 식물 생장 촉진 방법은 기생 선충 방제용 조성물, 식물 생장 촉진용 조성물 또는 미생물 제제를 식물의 종자, 잎, 줄기, 뿌리 또는 이에 근접한 토양에 직접 살포하거나 침지시키는 것일 수 있으며, 상기 기생 선충 방제용 조성물, 식물 생장 촉진용 조성물 또는 미생물 제제를 희석한 용액을 식물체 또는 이에 근접한 토양에 직접 살포하거나, 침지시킬 수 있으나, 이에 제한 되지 않는다. The method for controlling parasitic nematodes or promoting plant growth of the present invention may be spraying or immersing a composition for controlling parasitic nematodes, a composition for promoting plant growth, or a microbial agent directly on the seeds, leaves, stems, roots, or soil of a plant, or the soil adjacent thereto. , the composition for controlling parasitic nematodes, the composition for promoting plant growth, or a diluted solution of the microbial agent may be directly sprayed or immersed in the plant or the soil adjacent thereto, but is not limited thereto.
본 발명의 다른 하나의 양태는 기생 선충 방제용 조성물 또는 식물 생장 촉진용 미생물제제를 제공한다.Another aspect of the present invention provides a composition for controlling parasitic nematodes or a microbial agent for promoting plant growth.
상기 "기생 선충 방제", "식물 생장 촉진"은 전술한 바와 같다. The “parasitic nematode control” and “plant growth promotion” are the same as described above.
본 발명의 용어, "미생물 제제"는 미생물 자체를 직접 이용하거나 미생물을 포함한 제제를 이용하여 농작물에 피해를 주는 곤충, 응애, 선충 등의 해충과 각종 식물 병원균 또는 잡초를 효과적으로 방제하는데 쓰이는 생물 제제를 의미한다. The term "microbial agent" of the present invention refers to a biological agent used to effectively control pests such as insects, mites, nematodes, and various plant pathogens or weeds that damage crops by directly using the microorganisms themselves or using preparations containing microorganisms. it means.
상기 기생 선충 방제용 조성물, 식물 생장 촉진용 조성물 또는 미생물 제제는 농약학적으로 허용가능한 담체, 부형제 및/또는 첨가제를 추가로 포함할 수 있다. 상기 부형제는 농업 분야에서 통상적으로 사용되고 있는 것으로, 예를 들면, 규조토, 소석회 등의 산화물, 인회석 등의 인산염, 석고 등의 황산염, 클레이, 카올린, 벤토나이트, 산성백토, 석영, 실리카 등의 광물질 분말 등의 고체 담체와 충진제, 항응집제, 계면활성제, 유화제, 방부제, 가소제, 착색제, 증백제, 분산제, 유동화제 등을 추가하여 포함할 수 있다. 또한, 본 발명의 조성물 또는 제제를 식물에 처리하여 활성성분이 신속방출, 서방출, 지연된 방출할 수 있도록 당업계에서 공지된 방법을 사용하여 제형화할 수 있다. 제형화를 위해서는 통상적으로 사용하는 계면활성제, 희석제, 분산제, 보조제 등의 첨가제를 활성성분과 배합하여 수화제, 현탁제, 유제, 유탁제, 미탁제, 액제, 분산성 액제, 입상수화제, 입제, 분제, 액상수화제, 수면부상성입제, 정제, 훈증제, 도포제 등 각종 형태로 제제화하여 사용할 수 있다. 본 발명의 조성물을 제제화할 경우 사용 시기는 선충 발생 시 상시적으로 사용할 수 있으나, 사용 기간, 횟수 및 시기는 특별히 제한되지 않는다. The composition for controlling parasitic nematodes, the composition for promoting plant growth, or the microbial preparation may further include agrochemically acceptable carriers, excipients, and/or additives. The excipients are commonly used in the agricultural field, for example, oxides such as diatomaceous earth and slaked lime, phosphates such as apatite, sulfates such as gypsum, mineral powders such as clay, kaolin, bentonite, acid clay, quartz, and silica. It may include additional solid carriers, fillers, anti-coagulants, surfactants, emulsifiers, preservatives, plasticizers, colorants, brighteners, dispersants, fluidizers, etc. In addition, the composition or formulation of the present invention can be formulated using methods known in the art to treat plants to allow rapid, sustained, or delayed release of the active ingredient. For formulation, commonly used additives such as surfactants, diluents, dispersants, and auxiliaries are mixed with the active ingredients to create hydrating agents, suspensions, emulsions, emulsions, emulsifiers, liquids, dispersible liquids, granular hydrates, granules, powders, etc. It can be formulated and used in various forms such as liquid moisturizer, sleep aid, tablet, fumigant, and coating agent. When formulating the composition of the present invention, it can be used regularly when nematodes occur, but the period, number, and timing of use are not particularly limited.
또한, 본 발명의 조성물은 그 자체, 상기 제제 형태 또는 이들 제제를 추가로 희석하여 제조될 수 있는 사용형태로 사용할 수 있으며, 붓기, 분무, 연무(atomizing), 산포 또는 뿌리기(broadcasting)와 같은 통상적인 방법에 의해 사용될 수 있다.In addition, the composition of the present invention can be used as such, in the form of the above preparations, or in use forms that can be prepared by further diluting these preparations, and can be used in conventional ways such as pouring, spraying, atomizing, dispersing or broadcasting. It can be used by this method.
본 발명의 기생 선충 방제용 조성물, 식물 생장 촉진용 조성물 또는 미생물 제제는 공지된 다른 살선충제와 혼합하여 사용할 수 있다. 즉석 믹스(ready mix)의 형태로 사용할 수 있으며, 또는 살선충 활성 혼합물이 사용 시에 혼합되는 탱크 믹스와 같은 개별 제제 형태로 사용될 수 있다. 살진균제, 살충제, 살비제, 조류 기피제, 성장 물질, 식물 영양제, 및 토양 컨디셔너와 같은 다른 공지된 활성 혼합물과의 혼합도 가능하다. 특정 적용 목적을 위해, 특히 발아 후 방법에 적용되는 경우, 제제 중의 추가의 첨가제로서 식물-내약성 광유 또는 식물유(예를 들어, 시판 제품 "Rako Binol"또는 암모늄염, 예를 들면 황산암모늄 또는 티오황산암모늄을 함침시킬 수 있다.The composition for controlling parasitic nematodes, the composition for promoting plant growth, or the microbial preparation of the present invention can be used by mixing with other known nematicides. It can be used in the form of a ready mix, or in the form of individual preparations such as tank mixes into which the nematicidally active mixture is mixed at the time of use. Mixtures with other known active mixtures such as fungicides, insecticides, acaricides, bird repellents, growth substances, plant nutrients and soil conditioners are also possible. For specific application purposes, especially when applied in post-emergence methods, additional additives in the formulation may be used as plant-tolerated mineral oils or vegetable oils (e.g. the commercial product "Rako Binol") or ammonium salts, e.g. ammonium sulfate or ammonium thiosulfate. can be impregnated.
본 발명의 기생 선충 방제용 조성물, 식물 생장 촉진용 조성물 또는 미생물 제제는 살선충 유효량의 화합물 및 식물 생장 촉진제를 포함할 수 있으며, 상기 용어, "유효량"은 재배 식물 상에서 유충을 방제하는데 충분하거나 물질의 보호에 충분하고, 처리된 식물에 실질적인 손상을 초래하지 않는 조성물 또는 화합물의 양을 의미한다.The composition for controlling parasitic nematodes, the composition for promoting plant growth, or the microbial preparation of the present invention may include a nematicidal effective amount of a compound and a plant growth promoter, and the term "effective amount" refers to a substance or substance sufficient to control larvae on cultivated plants. means the amount of a composition or compound that is sufficient for the protection of and does not cause substantial damage to the treated plants.
본 발명의 살선충 유효량은 농약 제제당 사용량의 0.01 내지 100(mg/kg)일 수 있으나, 방제할 선충 종, 처리된 재배 식물 또는 물질, 기후 조건 및 사용되는 특정 화합물 등 다양한 요인들을 고려하여 유효량이 결정되는 것이므로, 이러한 점을 고려할 때 당분야의 통상적인 지식을 가진 자라면 상기 본 발명의 특정한 용도에 따른 적절한 유효량을 결정할 수 있을 것이다. The effective amount of nematodes of the present invention may be 0.01 to 100 (mg/kg) of the amount used per pesticide formulation, but the effective amount may be determined by taking into account various factors such as nematode species to be controlled, treated cultivated plants or materials, climatic conditions, and specific compounds used. Since this is determined, taking this into account, anyone with ordinary knowledge in the art will be able to determine an appropriate effective amount according to the specific use of the present invention.
본 발명의 버크홀데리아 JB-1 균주 또는 JB-2 균주, 상기 균주의 파쇄물, 상기 균주의 추출물, 상기 균주의 배양물, 상기 배양물의 농축액 및 상기 배양물의 건조물은 우수한 살선충 활성 및 식물의 생육 촉진 활성을 가지므로, 살선충 미생물 방제제에 대한 개발 및 생산에 있어 유용하게 활용되어, 친환경 농업의 구현뿐만 아니라, 농작물의 농약 잔류 및 토양 오염을 경감시켜 환경 보전에 기여할 수 있다.The Burkholderia JB-1 strain or JB-2 strain of the present invention, the lysate of the strain, the extract of the strain, the culture of the strain, the concentrate of the culture, and the dried product of the culture have excellent nematicidal activity and plant growth. Since it has a promoting activity, it can be usefully used in the development and production of nematode microbial control agents, and can contribute to environmental conservation by reducing pesticide residues and soil contamination in crops as well as implementing eco-friendly agriculture.
도 1는 버크홀데리아 속 (Burkholderia sp.) JB-1 및 JB-2 균주의 16s rRNA 염기서열을 버크홀데리아 속 공시균주와 비교하여 계통발생학적 모식도로 나타낸 결과이다.
도 2은 토양에서 분리된 버크홀데리아 속 (Burkholderia sp.) JB-1 및 JB-2 균주의 키틴 및 단백질 분해 활성을 나타낸 결과이다.
도 3은 다양한 버크홀데리아 속 (Burkholderia sp.) 균주 배양 추출물의 뿌리혹선충(Meloidogyne hispanica)에 대한 살선충 활성을 평가한 결과이다.
도 4는 버크홀데리아 속 (Burkholderia sp.) JB-1 및 JB-2 균주 배양 추출물의 자바니카 뿌리혹선충, 땅콩 뿌리혹선충, 고구마 뿌리혹선충, 당근 뿌리혹선충에 대한 살선충 활성을 평가한 결과이다.
도 5는 버크홀데리아 속 (Burkholderia sp.) JB-1 및 JB-2 균주 배양 추출물의 뿌리혹선충 외에 다른 기생 선충에 대한 살선충 활성을 평가한 결과이다.
도 6은 버크홀데리아 속 (Burkholderia sp.) JB-1 및 JB-2 균주 배양물의 포트 활성 (pot-test) 결과이다.
도 7은 버크홀데리아 속 (Burkholderia sp.) JB-1 및 JB-2 균주 배양물의 식물 방어 관련 유전자량 변화를 측정한 결과이다.
Figure 1 shows the results of a phylogenetic diagram comparing the 16s rRNA base sequences of Burkholderia sp. JB-1 and JB-2 strains with the Burkholderia genus reference strain.
Figure 2 shows the results showing the chitin and protein decomposition activities of Burkholderia sp. JB-1 and JB-2 strains isolated from soil.
Figure 3 shows the results of evaluating the nematicidal activity of culture extracts of various Burkholderia sp. strains against root-knot nematode (Meloidogyne hispanica).
Figure 4 shows the results of evaluating the nematicidal activity of culture extracts of Burkholderia sp. JB-1 and JB-2 strains against Javanica root-knot nematodes, peanut root-knot nematodes, sweet potato root-knot nematodes, and carrot root-knot nematodes.
Figure 5 shows the results of evaluating the nematicidal activity of culture extracts of Burkholderia sp. JB-1 and JB-2 strains against other parasitic nematodes in addition to root-knot nematodes.
Figure 6 shows the pot-test results of cultures of Burkholderia sp. JB-1 and JB-2 strains.
Figure 7 shows the results of measuring changes in plant defense-related genes in cultures of Burkholderia sp. JB-1 and JB-2 strains.
이하 본 발명을 실시예 및 실험예를 통하여 보다 상세하게 설명한다. 그러나 이들 실시예 및 실험예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예 및 실험예에 한정되는 것은 아니다. Hereinafter, the present invention will be described in more detail through examples and experimental examples. However, these examples and experimental examples are for illustrative purposes only and the scope of the present invention is not limited to these examples and experimental examples.
실시예 1. 토양으로부터 미생물 분리 및 동정Example 1. Isolation and identification of microorganisms from soil
전북 진안군 주천면 가마봉(35.980645, 127.406836)의 밭 토양 및 산림 토양에서 토양시료를 채취하였다. 채취한 토양을 대상으로 미생물을 분리하기 위하여 토양시료의 2배량의 Phosphate buffered saline(PBS, 0.8% NaCl, 0.02% KCl, 0.144% Na2HPO4; pH 7.4)를 넣고 1분 동안 균질화하여 원심분리 후 상층액을 분취하였다. 그 후 단계적으로 희석하여 감자 덱스트로스 고체 배지 (PDA; 4% potato starch, 20% dextrose, 15% agar)에 도말하여 30℃에서 2일간 배양 후 성장한 박테리아를 분리하였다. 분리한 박테리아는 그람 음성의 간균 형태이며 원형과 흰색 집락을 형성하였다. Soil samples were collected from field soil and forest soil in Gamabong (35.980645, 127.406836), Jucheon-myeon, Jinan-gun, Jeollabuk-do. To isolate microorganisms from the collected soil, Phosphate buffered saline (PBS, 0.8% NaCl, 0.02% KCl, 0.144% Na2HPO4; pH 7.4) was added twice as much as the soil sample, homogenized for 1 minute, centrifuged, and the supernatant was aliquoted. Afterwards, it was serially diluted, spread on potato dextrose solid medium (PDA; 4% potato starch, 20% dextrose, 15% agar), and grown at 30°C for 2 days to isolate the grown bacteria. The isolated bacteria were Gram-negative bacilli and formed round, white colonies.
박테리아의 동정을 위해 16s rRNA 유전자 염기서열 분석을 수행하였다. 구체적으로, 균주의 16s rRNA 유전자의 염기서열을 얻기 위하여 Qiagen사의 DNeasy Blood & Tissue kit을 이용하여 genomic DNA를 분리 하였고 이를 템플레이트로 사용하여 27F (서열번호 1), 1492R 프라이머 (서열번호 2)와 함께 PCR을 수행하였다. 증폭된 PCR 산물은 정제 후 (주)마크로젠에 의뢰하여 서열을 분석하였다. 염기서열은 NCBI의 BLAST을 이용하여 GenBank 데이터베이스의 염기서열과 비교하였다.16s rRNA gene sequence analysis was performed to identify bacteria. Specifically, in order to obtain the base sequence of the 16s rRNA gene of the strain, genomic DNA was isolated using Qiagen's DNeasy Blood & Tissue kit, and this was used as a template along with primers 27F (SEQ ID NO: 1) and 1492R (SEQ ID NO: 2). PCR was performed. The amplified PCR product was purified and then submitted to Macrogen Co., Ltd. for sequence analysis. The base sequence was compared with the base sequence in the GenBank database using NCBI's BLAST.
그 결과, 버크홀데리아 속 (Burkholderia sp.) JB-1 균주는 Burkholderia contaminans J2956 과 99.50% 유사도를 보였고, 상기 분리된 균주를 버크홀데리아 속 (Burkholderia sp.) JB-1로 명명하였다 (서열번호 3). 버크홀데리아 속 (Burkholderia sp.) JB-2 균주는 Burkholderia alba AD18 과 98.96% 유사도를 보여, 이에 따라 분리된 균주를 버크홀데리아 속 (Burkholderia sp.) JB-2로 명명하였다 (서열번호 4). 상기 JB-1 및 JB-2 균주는 한국생명공학연구원에 2022년 05월 17일자로 기탁하였다(수탁번호: KCTC14976BP, KCTC14976BP) (도 1).As a result, the Burkholderia sp. JB-1 strain showed 99.50% similarity to Burkholderia contaminans J2956, and the isolated strain was named Burkholderia sp. JB-1 (SEQ ID NO. 3). Burkholderia sp. JB-2 strain showed 98.96% similarity to Burkholderia alba AD18, and accordingly, the isolated strain was named Burkholderia sp. JB-2 (SEQ ID NO. 4) . The JB-1 and JB-2 strains were deposited with the Korea Research Institute of Bioscience and Biotechnology on May 17, 2022 (Accession Number: KCTC14976BP, KCTC14976BP) (Figure 1).
실시예 2. 버크홀데리아 속 (Burkholderia sp.) JB-1 및 JB-2 균주의 살선충 관련 효소 활성 비교Example 2. Comparison of nematode-related enzyme activities of Burkholderia sp. JB-1 and JB-2 strains
선충의 표피 및 알의 주된 성분인 단백질 및 키틴에 대한 버크홀데리아 속 (Burkholderia sp.) JB-1 및 JB-2 균주의 분해 활성을 확인하기 위하여, 각 효소와 반응할 기질을 함유하고 있는 고체 선별배지를 사용하였다. To confirm the decomposition activity of Burkholderia sp. JB-1 and JB-2 strains on protein and chitin, which are the main components of nematode epidermis and eggs, solids containing substrates to react with each enzyme were used. A selection medium was used.
키틴 분해 활성은 2% Colloidal chitin, 단백질 분해 활성은 2% 스킴 밀크(skim milk)를 각각 R2A 배지(R2A; 0.05% Proteose peptone, 0.05% Casamino acids, 0.05% Yeast extract, 0.05% Dextrose, 0.05% Soluble starch, 0.03% Dipotassium phosphate, 0.005% Magnesium sulfate, 0.03% Sodium pyruvate, 1.5% Agar)에 첨가하여 선별배지를 제조하였다. 각 선별배지에 선발한 균주를 접종하여 30℃에서 48시간 동안 배양한 후 투명환 형성 유무에 따라 기질 특이적 효소 분해능 여부를 확인하였다. 2% Colloidal chitin for chitinolytic activity and 2% skim milk for proteolytic activity were added to R2A medium (R2A; 0.05% Proteose peptone, 0.05% Casamino acids, 0.05% Yeast extract, 0.05% Dextrose, 0.05% Soluble). A selection medium was prepared by adding starch, 0.03% Dipotassium phosphate, 0.005% Magnesium sulfate, 0.03% Sodium pyruvate, and 1.5% Agar). The selected strains were inoculated into each selection medium and cultured at 30°C for 48 hours, and then the substrate-specific enzymatic decomposition ability was checked based on the presence or absence of transparent ring formation.
그 결과, 두 종의 버크홀데리아 균주 접종 시 2종의 기질에 대하여 모두 투명환이 선명하게 형성됨으로써 살선충 활성이 우수한 것을 확인하였다 (도 2).As a result, it was confirmed that when two types of Burkholderia strains were inoculated, transparent rings were clearly formed on both types of substrates, showing excellent nematicidal activity (Figure 2).
실시예 3. 버크홀데리아 속 (Burkholderia sp.) JB-1 및 JB-2 균주 추출물의 뿌리혹선충에 대한 활성 검정Example 3. Activity assay of extracts from Burkholderia sp. JB-1 and JB-2 strains against root-knot nematodes
실시예 3-1. 버크홀데리아 속 균주 추출물의 뿌리혹선충에 대한 활성 검정Example 3-1. Activity test of Burkholderia strain extract against root-knot nematodes
뿌리혹선충(Meloidogyne hispanica)이 감염된 토양시료 300g을 약 5 리터의 물에 넣고 섞어 만든 흙탕물을 250㎛ 구멍 크기의 채로 거른 다음, 얻어진 흙탕물을 45㎛ 구멍 크기의 채로 걸러 선충을 모았다. 채에 걸린 선충을 물로 회수하고, 회수한 선충이 포함된 약간의 흙탕물을 킴와이프(Kimwipe)를 깔은 채에 붓고 적당량의 물을 채워 24시간 동안 정치한 후, 채를 통과한 물에 모여있는 선충을 다시 45㎛ 구멍 크기의 채로 걸러 회수하여 살선충 활성 조사에 이용하였다.300 g of soil sample infected with root-knot nematode ( Meloidogyne hispanica ) was mixed with about 5 liters of water, the muddy water was filtered through a pore size of 250㎛, and the resulting muddy water was filtered through a pore size of 45㎛ to collect nematodes. Recover the nematodes caught in the strainer with water, pour some muddy water containing the recovered nematodes into a strainer lined with a Kimwipe, fill it with an appropriate amount of water, let it stand for 24 hours, and then collect the collected nematodes in the water that passed through the strainer. The nematodes were filtered and recovered with 45㎛ pore size and used to investigate nematicidal activity.
JB-1 및 JB-2를 포함한 버크홀데리아 속 (Burkholderia sp.) 균주를 각각 LB 액체 배지(10% Yeast extract, 5% Tryptone, 10% Sodium chloride)에 접종하고 30℃에서 180 rpm으로 48시간 배양 후 원심 분리하였다. 수득한 상층액을 동량의 에틸 아세테이트로 추출하여 감압 농축하고 이를 메탄올로 용해하였다. Burkholderia sp. strains, including JB-1 and JB-2, were each inoculated into LB liquid medium (10% Yeast extract, 5% Tryptone, 10% Sodium chloride) and incubated at 30°C at 180 rpm for 48 hours. After culturing, it was centrifuged. The obtained supernatant was extracted with an equal amount of ethyl acetate, concentrated under reduced pressure, and dissolved in methanol.
96well-plate에 상기 회수한 뿌리혹선충 (Meloidogyne hispanica)을 95㎕ 처리하고, 이에 버크홀데리아 속 균주 추출물을 0.5 ㎎/mL 농도가 되도록 처리 하였다. 대조구로는 선충탄(5% fosthiazate) 희석액을 처리해주었으며, 무처리구로 메탄올을 처리하여 총 볼륨을 100 ㎕ 로 맞춰주었다. 25℃에서 보관하여 48시간 이후 Abbott's formula를 활용하여 살선충율을 조사하였다.95㎕ of the recovered root-knot nematode ( Meloidogyne hispanica ) was treated in a 96 well-plate, and the Burkholderia genus strain extract was treated to a concentration of 0.5 mg/mL. As a control group, a diluted solution of nematode charcoal (5% fosthiazate) was treated, and as an untreated group, methanol was treated to adjust the total volume to 100 ㎕. It was stored at 25°C and the nematode rate was investigated using Abbott's formula after 48 hours.
살선충율(%)= (처리구의 사망률-무처리구의 사망률) / 100-무처리구 사망률 Х 100Nematode rate (%) = (mortality rate of treated group - mortality rate of untreated group) / 100 - mortality rate of untreated group Х 100
그 결과, 버크홀데리아 속 (Burkholderia sp.) JB-1 및 JB-2 균주는 버크홀데리아 속의 다른 균주보다 약 10% 내지 약 85%이상의 우수한 살선충 활성을 가지는 것을 확인하였으며, 살선충제인 선충탄과 유사하거나, 이보다 약 10%이상 높은 살선충 활성을 가지는 것을 확인하였다 (도 3). As a result, it was confirmed that Burkholderia sp. JB-1 and JB-2 strains had about 10% to about 85% more excellent nematicidal activity than other strains of the Burkholderia genus, and that they were effective against nematodes, which are nematodes. It was confirmed that it had a nematicidal activity similar to or about 10% higher than that of Tan (Figure 3).
실시예 3-2. 버크홀데리아 속 JB-1 및 JB-2 균주 추출물의 뿌리혹선충에 대한 활성 검정Example 3-2. Activity assay of extracts from strains of Burkholderia genus JB-1 and JB-2 against root-knot nematodes
실시예 3-1.에서 실시한 실험방법과 동일 방법으로, 자바니카 뿌리혹선충 (Meloidogyne javanica), 땅콩 뿌리혹선충 (Meloidogyne arenaria), 고구마 뿌리혹선충 (Meloidogyne incognita), 당근 뿌리혹선충 (Meloidogyne hapla)을 회수하여, 실시예 3-1.에서 가장 효과가 좋았던 버크홀데리아 속 JB-1 및 JB-2 균주 추출물의 살선충 활성(살선충율)을 비교하였다. By the same method as the experimental method performed in Example 3-1, Javanica root-knot nematode ( Meloidogyne javanica ), peanut root-knot nematode ( Meloidogyne arenaria ), sweet potato root-knot nematode ( Meloidogyne incognita ), and carrot root-knot nematode ( Meloidogyne hapla ) were recovered. , the nematicidal activity (nematocide rate) of the extracts of Burkholderia genus JB-1 and JB-2 strains, which were the most effective in Example 3-1, were compared.
그 결과, Meloidogyne hispanica 외에 자바니카 뿌리혹선충, 땅콩 뿌리혹선충, 고구마 뿌리혹선충, 당근 뿌리혹선충와 같은 일반적인 뿌리혹선충에 대하여도 버크홀데리아 속 JB-1 및 JB-2 균주 추출물은 대조군과 유사하거나, 이보다 우수한 살선충 활성을 가지는 것을 확인하였다 (도 4). As a result, the extracts of Burkholderia genus JB-1 and JB-2 strains were similar to or better than the control group for common root-knot nematodes such as Javanica root-knot nematode, peanut root-knot nematode, sweet potato root-knot nematode, and carrot root-knot nematode, in addition to Meloidogyne hispanica. It was confirmed to have nematicidal activity (Figure 4).
실시예 3-3. 버크홀데리아 속 JB-1 및 JB-2 균주 추출물 다양한 기생 선충에 대한 활성 검정Example 3-3. Burkholderia genus JB-1 and JB-2 strain extract activity assay against various parasitic nematodes
실시예 3-1.에서 실시한 실험방법과 동일 방법으로, 뿌리혹선충 외에 주요 식물 기생 선충인 줄기선충류, 잎선충류, 씨스트선충류에 대한 버크홀데리아 속 JB-1 및 JB-2 균주 추출물의 살선충 활성(살선충율)을 비교하기 위하여, 대표적으로, Meloidogyne hispanica, Ditylenchus destructor(줄기선충), Aphelenchoides subtenuis(잎선충), Heterodera trifolii(씨스트선충)을 선정하였다. In the same manner as the experiment performed in Example 3-1, the nematode kill of extracts from Burkholderia strains JB-1 and JB-2 against stem nematodes, leaf nematodes, and cyst nematodes, which are major plant parasitic nematodes in addition to root-knot nematodes. To compare the activity (nematodesis rate), Meloidogyne hispanica, Ditylenchus destructor (stem nematode), Aphelenchoides subtenuis (leaf nematode), and Heterodera trifolii (cyst nematode) were selected as representative examples.
그 결과, 버크홀데리아 속 (Burkholderia sp.) JB-1 및 JB-2 균주 추출물은 0.5 mg/mL의 농도에서 Meloidogyne hispanica에서는 69.5 % 및 79.0 %, Ditylenchus destructor(줄기선충)에서는 84.2 % 및 91.3 %, Aphelenchoides subtenuis(잎선충)에서는 79.8 % 및 90.2 %, Heterodera trifolii(씨스트선충)에서는 61.9 % 및 74.0 %의 살선충 효율을 보여 뿌리혹선충 외에 주요 식물 기생 선충에 대해서도 우수한 살선충 활성을 가지는 것을 확인하였다 (도 5).As a result, Burkholderia sp. JB-1 and JB-2 strain extracts reduced 69.5% and 79.0% of Meloidogyne hispanica and 84.2% and 91.3% of Ditylenchus destructor (stem nematode) at a concentration of 0.5 mg/mL. , nematicidal efficiencies of 79.8% and 90.2% for Aphelenchoides subtenuis (leaf nematode) and 61.9% and 74.0% for Heterodera trifolii (cyst nematode), confirming that it has excellent nematicidal activity not only against root-knot nematodes but also against major plant parasitic nematodes. (Figure 5).
실시예 4. 버크홀데리아 속 (Burkholderia sp.) JB-1 및 JB-2 균주 배양물의 활성 검정Example 4. Activity assay of Burkholderia sp. JB-1 and JB-2 strain cultures
실시예 4-1. 버크홀데리아 속 (Burkholderia sp.) JB-1 및 JB-2 균주 배양물의 포트 활성(pot-test) 검정Example 4-1. Pot-test assay of Burkholderia sp. JB-1 and JB-2 strain cultures
버크홀데리아 속 (Burkholderia sp.) JB-1, JB-2 균주 배양물에 대한 뿌리혹선충의 방제 효과를 in vivo 수준에서 확인 평가하고자 하기와 같은 방법으로 포트 활성을 검정하였다. To confirm and evaluate the control effect of root-knot nematodes on cultures of Burkholderia sp. JB-1 and JB-2 strains at the in vivo level, pot activity was tested in the same manner as follows.
뿌리혹선충 포트 실험은 부산대학교 밀양캠퍼스 첨단온실에서 실시하였다. 500g의 뿌리혹선충이 감염된 토양에 상기 균주를 LB 액체 배지에서 접종하고 30℃에서 180 rpm으로 48시간 배양 후 오토클레이브에서 121℃ 15분간 멸균시킨 배양물 50mL를 처리 후 잘 섞어주었다. 대조구로는 선충탄(5% fosthiazate) 희석액 또는 버크홀데리아 리노젠시스(Burkholderia rinojensis) 배양물을 처리하였다. 그 후 7일 후 3주간 재배한 Rutgers 품종의 토마토를 모종해주었다. 45일 이후 토마토의 뿌리를 수돗물로 깨끗이 씻은 후 난낭 수 및 식물 성장을 조사하였다. The root-knot nematode pot experiment was conducted in a state-of-the-art greenhouse at the Miryang Campus of Pusan National University. The above strain was inoculated in LB liquid medium into soil infected with 500 g of root-knot nematodes, cultured for 48 hours at 30°C at 180 rpm, and then treated with 50mL of the culture sterilized in an autoclave at 121°C for 15 minutes and mixed well. As a control, nematode charcoal (5% fosthiazate) dilution or Burkholderia rinojensis culture was treated. Seven days later, tomato seedlings of the Rutgers variety, which had been grown for three weeks, were planted. After 45 days, the roots of the tomatoes were washed thoroughly with tap water and the number of egg cysts and plant growth were examined.
그 결과, JB-1 및 JB-2 균주 배양물의 처리구에서 대조구보다 지상부 길이가 현저히 증가함을 확인하였으며(도 6A, 6B, 6C), 지상부 무게는 무처리 대비 약 3.6-7.6배 증가, 선충탄 처리구 대비 약 1.8-3.8배 증가, B. rinojensis 처리구 대비 약 4.5-9.5배 증가하였고, 뿌리 무게 또한 무처리, 선충탄 처리구, 및 B. rinojensis 처리구 대비 증가하여, JB-1 및 JB-2 균주 배양물의 증가 작물 생육 촉진 효과를 확인하였다 (도 6D). As a result, it was confirmed that the length of the aerial part significantly increased in the treatment group of JB-1 and JB-2 strain cultures compared to the control group (Figures 6A, 6B, 6C), and the weight of the aerial part increased by about 3.6-7.6 times compared to the untreated group, nematode charcoal. It increased approximately 1.8-3.8 times compared to the treatment group, increased approximately 4.5-9.5 times compared to the B. rinojensis treatment group, and root weight also increased compared to the untreated, nematode charcoal treated, and B. rinojensis treated groups, cultivating strains of JB-1 and JB-2. The effect of increasing water on promoting crop growth was confirmed (Figure 6D).
또한, 뿌리 무게 당 난낭 수의 경우, JB-1 및 JB-2 균주 배양물의 처리구가 무처리 및 B. rinojensis 처리구보다 적게 나타났으며, 양성대조구인 선충탄과 유사한 수준인 것을 확인함으로써, JB-1 및 JB-2 균주 배양물의 뿌리혹선충 억제에 효과가 있음을 확인하였다 (도 6E).In addition, the number of egg cysts per root weight was lower in the treated groups of JB-1 and JB-2 strain cultures than the untreated and B. rinojensis treated groups, and was confirmed to be at a similar level to the nematode charcoal, which is a positive control, confirming that JB- It was confirmed that the cultures of strains 1 and JB-2 were effective in suppressing root-knot nematodes (Figure 6E).
실시예 4-2. 버크홀데리아 속 (Burkholderia sp.) JB-1 및 JB-2 균주 배양물의 식물 방어 관련 유전자량 측정Example 4-2. Measurement of plant defense-related genes in cultures of Burkholderia sp. JB-1 and JB-2 strains.
실시예 4-1.의 버크홀데리아 속 (Burkholderia sp.) JB-1, JB-2 균주 배양물을 처리한 Rutgers 품종의 토마토의 뿌리를 채취하여 액체질소에 넣어 동결시켰다. 동결된 뿌리를 곱게 갈아준 후 QIAGEN사의 RNeasy Plant Mini Kit를 사용하여 mRNA를 추출하였으며, Thermo사의 High-Capacity cDNA Reverse Transcription Kit를 사용하여 추출한 mRNA를 cDNA로 역전사하였다. 그 다음, 식물의 방어 관련 유전자의 Primer인 SlPR1, SlLOX1, SpWRKY1, CDPK15, RbohB와 Thermo사의 SYBR?? Green Master Mix™를 사용하여 qPCR을 진행하였고, 비교 Ct△△Ct 방법을 사용하여 유전자 발현의 배수 변화를 계산하였다.The roots of tomatoes of the Rutgers variety treated with the Burkholderia sp. JB-1 and JB-2 strain cultures of Example 4-1 were collected and frozen in liquid nitrogen. After grinding the frozen roots finely, mRNA was extracted using QIAGEN's RNeasy Plant Mini Kit, and the extracted mRNA was reverse transcribed into cDNA using Thermo's High-Capacity cDNA Reverse Transcription Kit. Next, primers for plant defense-related genes, SlPR1, SlLOX1, SpWRKY1, CDPK15, RbohB and Thermo's SYBR?? qPCR was performed using Green Master Mix™, and the fold change in gene expression was calculated using the comparative Ct△△Ct method.
그 결과, JB-1, JB-2 균주 배양물을 처리시 식물의 방어 관련 유전자인 SlPR1, SlLOX1 유전자 발현량이 무처리 대비 증가함을 확인하였으며, 이를 통해 살리실산(SA) 및 자스몬산(JA) 관련된 방어 유전자가 활성화되었음을 알 수 있었으며, SpWRKY1, CDPK15, RbohB 유전자 발현량이 무처리 대비 증가함을 확인하였고, 이를 통해 활성산소(ROS) 관련 유전자가 활성화되었음을 알 수 있었다. As a result, it was confirmed that when treating JB-1 and JB-2 strain cultures, the expression level of SlPR1 and SlLOX1 genes, which are plant defense-related genes, increased compared to untreated, and through this, salicylic acid (SA) and jasmonic acid (JA)-related It was confirmed that defense genes were activated, and the gene expression levels of SpWRKY1, CDPK15, and RbohB were confirmed to increase compared to untreated, which showed that reactive oxygen species (ROS)-related genes were activated.
상기와 같이, JB-1, JB-2 균주 배양물을 식물에 처리한 경우, 무처리군에 비해 식물 방어 관련 유전자가 높게 발현되어 선충에 대한 식물 방어를 유도하였음을 알 수 있었고, JB-1, JB-2 균주 배양물은 살선충 활성뿐만이 아니라 식물에도 작용하여 선충 방제에 대한 도움이 될 수 있음을 확인하였다 (도 7).As described above, when plants were treated with JB-1 and JB-2 strain cultures, plant defense-related genes were expressed at a higher level compared to the untreated group, indicating that plant defense against nematodes was induced, and JB-1 , it was confirmed that the JB-2 strain culture not only has nematicidal activity but also acts on plants, which can be helpful in controlling nematodes (Figure 7).
이상의 설명으로부터, 본 발명이 속하는 기술분야의 당업자는 본 발명이 그 기술적 사상이나 필수적 특징을 변경하지 않고서 다른 구체적인 형태로 실시될 수 있다는 것을 이해할 수 있다. 이와 관련하여, 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적인 것이 아닌 것으로 이해해야만 한다. 본 발명의 범위는 상기 상세한 설명보다는 후술하는 특허 청구범위의 의미 및 범위 그리고 그 등가 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다. From the above description, those skilled in the art to which the present invention pertains can understand that the present invention can be implemented in other specific forms without changing its technical idea or essential features. In this regard, the embodiments described above should be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed as including the meaning and scope of the patent claims described below rather than the detailed description above, and all changes or modified forms derived from the equivalent concept thereof are included in the scope of the present invention.
Claims (11)
Burkholderia JB-1 strain deposited with accession number KCTC14975BP or Burkholderia JB-2 strain deposited with accession number KCTC14976BP.
The strain according to claim 1, wherein the strain has nematicidal activity.
The strain according to claim 1, wherein the strain has growth promoting activity.
The strain according to claim 1, wherein the strain has chitin and proteolytic activity.
A parasite comprising one or more selected from the group consisting of the strain of any one of claims 1 to 4, lysate of the strain, extract of the strain, culture of the strain, concentrate of the culture, and dried material of the culture. Composition for controlling nematodes.
The method of claim 5, wherein the parasitic nematodes include sweet potato root-knot nematodes (Meloidogyne incognita), peanut root-knot nematodes (M. arenaria), carrot root-knot nematodes (M. hapla), Javanica root-knot nematodes (M. javanica), and leaf nematodes (Aphelenchodies). ), one or more nematodes selected from the group consisting of Ditylenchus, Globodera, Heterodera, Pratylenchus, Trichodorus, and Xiphinema, for controlling parasitic nematodes Composition.
A method for controlling parasitic nematodes, comprising treating plants or parasitic nematodes with the composition for controlling parasitic nematodes of claim 5.
The method of claim 7, wherein the composition is directly sprayed or immersed in the seeds, leaves, stems, roots of plants, or soil adjacent thereto.
Contains as an active ingredient at least one selected from the group consisting of the strain of any one of claims 1 to 4, lysate of the strain, extract of the strain, culture of the strain, concentrate of the culture, and dried material of the culture. A composition for promoting plant growth.
A microbial preparation comprising the composition of claim 5 or 9.
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| KR1020220096930A KR20240020308A (en) | 2022-08-03 | 2022-08-03 | Novel Burkholderia sp. having nematicidal activity and use thereof |
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| KR20240020308A true KR20240020308A (en) | 2024-02-15 |
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| KR1020220096930A KR20240020308A (en) | 2022-08-03 | 2022-08-03 | Novel Burkholderia sp. having nematicidal activity and use thereof |
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