KR20220162899A - Microbacterium testaceum DDP398 having activities of promoting plant growth and controlling plant disease, and uses thereof - Google Patents
Microbacterium testaceum DDP398 having activities of promoting plant growth and controlling plant disease, and uses thereof Download PDFInfo
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- KR20220162899A KR20220162899A KR1020210070543A KR20210070543A KR20220162899A KR 20220162899 A KR20220162899 A KR 20220162899A KR 1020210070543 A KR1020210070543 A KR 1020210070543A KR 20210070543 A KR20210070543 A KR 20210070543A KR 20220162899 A KR20220162899 A KR 20220162899A
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Abstract
Description
식물 생장 촉진 및 식물병 방제 활성을 갖는 마이크로박테리움 테스타세움 (Microbacterium testaceum) DDP398 균주 및 이의 용도에 관한 것이다.It relates to a Microbacterium testaceum DDP398 strain having plant growth promoting and plant disease control activities and uses thereof.
현재 농업에 사용되는 화학비료와 무기질비료의 사용을 줄여 토양과 환경을 보호하면서 작물 재배를 활성화할 미생물 제제를 찾는 것은 중요한 의미가 있다. 근권에 존재하는 유용 미생물은 환경 오염의 주요인으로 뽑히는 화학비료의 대체제로서 주목을 받아왔으며, 식물 생장 촉진 미생물로 Pseudomonas, Burkholderia, Bacillus, Enterobacter, Arthrobacter, Acinetobacter 속 등이 연구되어왔다. 식물 뿌리는 토양 근권에 존재하는 유용 미생물의 성장을 촉진하는 저분자 탄소원을 방출하고, 토양 근권에서 존재하는 유용 미생물은 질소 고정, 미네랄 고정 및 가용화를 통해 식물에 영양분을 제공하거나 생장호르몬인 indole-3-acetic acid (IAA) 및 siderophore 생성, 항생물질, 세포외 효소 등을 제공하여 식물 생장을 촉진하며 식물 병원성 곰팡이의 생장을 억제해 식물 생장에 도움을 준다. It is important to find a microbial agent that will activate crop cultivation while protecting the soil and environment by reducing the use of chemical and inorganic fertilizers currently used in agriculture. Useful microorganisms existing in the rhizosphere have been attracting attention as substitutes for chemical fertilizers, which are the main cause of environmental pollution, and Pseudomonas, Burkholderia, Bacillus, Enterobacter, Arthrobacter, and Acinetobacter genera have been studied as plant growth promoting microorganisms. Plant roots release low-molecular-weight carbon sources that promote the growth of useful microorganisms in the soil rhizosphere, and useful microorganisms in the soil rhizosphere provide nutrients to plants through nitrogen fixation, mineral fixation, and solubilization, or indole-3, a growth hormone. -Promotes plant growth by providing acetic acid (IAA) and siderophore generation, antibiotics, and extracellular enzymes, and helps plant growth by inhibiting the growth of plant pathogenic fungi.
식물 성장 촉진 근권세균(Plant growth promoting rhizobacteria, PGPR)은 식물의 근권에 존재하면서 식물에 성장을 촉진하는 토양 미생물로, 식물의 성장과 발달에 직·간접적으로 도움을 준다. Plant growth promoting rhizobacteria (PGPR) are soil microorganisms that exist in the rhizosphere of plants and promote plant growth, directly or indirectly helping plants grow and develop.
인산은 식물의 광합성 작용, 에너지 전달, 신호전달, 고분자물질의 생합성 등 모든 주요 대사 과정에서 중요한 역할을 하는 필수영양소이며, 근권세균(PGPR) 균주는 인산을 가용화하여 식물 성장에 직접적으로 도움을 줄 수 있다. Phosphoric acid is an essential nutrient that plays an important role in all major metabolic processes such as photosynthesis, energy transmission, signal transduction, and biosynthesis of macromolecules in plants. can
또한, 대기 중의 질소는 생물체가 이용할 수 없는 형태로 존재하지만 PGPR 균주는 이를 생물체가 이용할 수 있는 질산염 형태로 변화시켜 질소원을 제공함으로써 식물 성장에 도움을 준다. In addition, nitrogen in the atmosphere exists in a form that cannot be used by living things, but the PGPR strain converts it into a form of nitrate that can be used by living things and provides a nitrogen source to help plant growth.
식물은 성장과 발달을 위해 다양한 영양소를 필요하며, 토양, 물, 공기로부터 필수 영양성분을 획득한다. 토양 유래 다량 영양소로는 N, P, K, S, Ca 및 Mg 등이 있으며, 미량 영양소로는 B, Cl, Cu, Fe, Mn, Mo, Zn 및 Ni 등이 있다. 그러나, 대부분 식물이 이용하기 어려운 불용성 형태로 존재하고 있어 이용하는데 어려움이 있다. Plants require a variety of nutrients for growth and development, and obtain essential nutrients from soil, water, and air. Soil-derived macronutrients include N, P, K, S, Ca, and Mg, and micronutrients include B, Cl, Cu, Fe, Mn, Mo, Zn, and Ni. However, it is difficult to use because it exists in an insoluble form that is difficult for most plants to use.
미생물은 인산, 규소, 아연과 같은 광물을 가용화하는데 중요한 역할을 하는 것으로 알려져 있으며, 미생물이 생산하는 유기산에 의해 다양한 불용성 광물들(mineral)이 가용화 되어짐에 따라 식물의 이용도(availability)를 증가시킨다.Microorganisms are known to play an important role in solubilizing minerals such as phosphoric acid, silicon, and zinc, and as various insoluble minerals are solubilized by organic acids produced by microorganisms, the availability of plants increases. .
인산은 식물의 핵심 영양요소이면서 식물의 광합성 작용, 에너지 전달, 신호 전달, 고분자물질의 생합성 등 모든 주요 대사 과정에서 중요한 역할을 하는 것으로 알려져 있다. 토양 중에 약 0.05%(w/w)를 차지하나 대부분 불용성 형태로 존재하여 이용하는데 어려움이 있다. 근권에 존재하는 인산 가용화균은 토양 속에 존재하는 인을 가용화 및 미네랄화하여 유기물과 무기물로부터 효과적으로 인을 방출하며, 주로 HPO4 2-, H2PO4-형태로 식물이 이용할 수 있도록 도와준다.Phosphoric acid is a key nutrient for plants and is known to play an important role in all major metabolic processes such as photosynthesis, energy transmission, signal transmission, and biosynthesis of macromolecules. It occupies about 0.05% (w/w) in soil, but it is difficult to use because most of them exist in insoluble form. Phosphorus-solubilizing bacteria present in the rhizosphere solubilize and mineralize phosphorus present in the soil, effectively release phosphorus from organic and inorganic matter, and help plants use it mainly in the form of HPO 4 2- and H 2 PO 4- .
지각의 평균칼슘함량은 약 3.6%로 다른 식물영양원소보다 비교적 많다. 칼슘(Ca)은 회장석, 사장석, 각섬석, 녹섬석 등과 같은 1차 광물에 주로 존재하고 있으며 토양 중의 칼슘함량은 모암의 종류와 풍화 정도에 따라 달라지지만 대개 0.1~5%이며, 특히 석회암을 모암으로 하여 발달한 토양에 많다. 칼슘은 Ca2+의 형태로 흡수되어 식물의 조직 내에서 Ca2+ 형태로 존재하는데, 주로 세포벽에 다량 존재하며, 펙틴 등 세포벽 구성물질의 COO-기에 결합하여 세포벽의 구조적인 안정성을 높여 주고 세포막에서도 이러한 역할을 한다고 알려져 있다. The average calcium content of the earth's crust is about 3.6%, which is relatively more than other plant nutrients. Calcium (Ca) is mainly present in primary minerals such as plagioclase, plagioclase, amphibole, and oleophobite. As a result, there are many in the developed soil. Calcium is absorbed in the form of Ca 2+ and exists in the form of Ca 2+ in plant tissues. It is mainly present in cell walls in large quantities. It is also known to play this role.
일반적으로 작물체 중 칼슘이 결핍되는 경우 막의 투과성을 손상시켜 세포에 있는 확산성을 갖는 물질의 보존을 어렵게 하여 막으로부터 누출되고, 최종적으로 막 구조의 손상을 야기할 수 있다. In general, when calcium is deficient in crop plants, the permeability of the membrane is impaired, making it difficult to preserve the diffusible material in the cells, leaking from the membrane, and finally damaging the membrane structure.
방해석(Calcite)의 가용화는 미생물이 생산하는 유기산, 무기산, 킬레이트 물질 및 세포 외 다당류의 생산에 의한 산성화와 같은 다양한 메커니즘에 의해 이루어지며, 그중에서도 유기산 생산이 방해석을 가용화하는데 가장 효과적인 방법이다.Solubilization of calcite is achieved by various mechanisms such as acidification by the production of organic acids, inorganic acids, chelating substances, and extracellular polysaccharides produced by microorganisms. Among them, organic acid production is the most effective method for solubilizing calcite.
규소(Silicon)는 지구상에서 두 번째로 풍부한 원소(27%)로서 일반적으로 알루미늄(Al), 마그네슘(Mg), 칼슘(Ca), 나트륨(Na), 칼륨(K), 철(Fe)과 불용성의 실리케이트(SiO3) 형태로 존재하고 있다. Silicon is the second most abundant element (27%) on Earth and is generally insoluble with aluminum (Al), magnesium (Mg), calcium (Ca), sodium (Na), potassium (K) and iron (Fe). It exists in the form of silicate (SiO 3 ).
규소는 아직 식물 성장에 필수적인 요소로 인식되지 않지만, 식물의 성장, 발달, 수확량 및 질병 저항성에 미치는 효과는 다양한 식물 종에서 관찰되고 있다. 또한, 생물적 및 비생물적 스트레스를 견딜 수 있는 잠재력을 제공하며, 곤충 및 기타 해충의 공격으로부터 질병에 대한 저항성을 향상시키는 효과가 있다. 그러나 식물은 풍화에 의해 방출되거나 토양수(Si(OH)4의 형태로 흡수가능)에 용해되지 않는 한 Si를 직접적으로 이용이 불가능하다. Although silicon is not yet recognized as an essential element for plant growth, its effects on plant growth, development, yield and disease resistance have been observed in a variety of plant species. In addition, it provides the potential to withstand biotic and abiotic stresses, and has the effect of improving resistance to disease from attack by insects and other pests. However, plants cannot use Si directly unless it is released by weathering or dissolved in soil water (which can be absorbed in the form of Si(OH) 4 ).
따라서, 규소 이용률을 높이기 위해서 규소 가용화능을 가진 균주를 이용하여 식물이 이용 가능한 형태로 바꿔 흡수를 용이하게 도와준다. 토양 속에 많은 미생물이 존재하지만 규소 가용화균은 거의 존재하지 않으며, 주로 Al2SiO5와 같은 규산염을 분해할 수 있다. 또한, 규소 가용화 균주는 불용성 형태의 규소뿐만 아니라 칼륨 및 인산염을 용해시키는 데 중요한 역할을 하여 토양 비옥도를 높이고 수확 생산성을 향상시킬 수 있다.Therefore, in order to increase the utilization rate of silicon, strains having silicon solubilizing ability are used to convert the silicon into a form usable by plants, thereby facilitating absorption. There are many microorganisms in the soil, but few silicon-solubilizing bacteria exist, and they can mainly decompose silicates such as Al 2 SiO 5 . In addition, silicon-solubilizing strains play an important role in dissolving insoluble forms of silicon as well as potassium and phosphate, which can increase soil fertility and improve harvest productivity.
아연은 식물 성장을 위해 필수적인 미량영양소로 식물체에는 탄수화물 대사, Auxin 대사에 관여하며, 상당한 항산화 작용을 하는 것으로 알려져 있다. 식물체에서 아연 결핍은 성장발달을 지연, 황화 현상 및 잎의 크기가 감소되며, 생산량, 꽃가루 형성, 뿌리 발달, 수분 섭취 및 운송 등뿐만 아니라 열, 빛, 곰팡이 감염에 대한 민감성에 영향을 미친다. 식물은 divalent cation(2가 양이온)의 형태로 아연을 흡수할 수 있지만 극히 적은 양이 수용성 형태로 토양 속에 존재하며, 대부분 불용성 복합체와 광물의 형태로 존재한다. 아연 가용화균은 아연을 공급할 수 있는 잠재적인 대안 방법이며, 불용성 아연을 이용할 수 있는 형태로 바꿔 줌으로써 식물의 성장과 발달을 향상시킬 수 있다.Zinc is an essential micronutrient for plant growth, and is involved in carbohydrate metabolism and Auxin metabolism in plants, and is known to have a significant antioxidant effect. Zinc deficiency in plants causes delayed growth, yellowing and reduced leaf size, and affects yield, pollen formation, root development, water intake and transport, as well as susceptibility to heat, light and fungal infection. Plants can absorb zinc in the form of divalent cations, but only a small amount is present in soil in water-soluble form, mostly in the form of insoluble complexes and minerals. Zinc-solubilizing bacteria are a potential alternative method of supplying zinc and can improve plant growth and development by converting insoluble zinc to a usable form.
한편, 사이드로포어(siderophore)는 식물 성장에 필수 원소인 철 이온에 특이적으로 결합하는 물질로 직접적으로 식물 성장에 도움을 주는 물질이면서 식물병원균이 철 이온을 흡수하는 것을 경쟁적으로 저해함으로써 식물병원균의 생육을 억제할 수 있는 물질이다. 따라서, PGPR 균주는 사이드로포어를 생성함으로써 식물 성장에 도움을 줌과 동시에 식물병 방제 효과를 나타낼 수도 있다. On the other hand, siderophore is a substance that specifically binds to iron ion, which is an essential element for plant growth. It is a substance that directly helps plant growth and competitively inhibits plant pathogens from absorbing iron ions. A substance that can inhibit the growth of Therefore, the PGPR strain may help plant growth and exhibit a plant disease control effect by generating siderophores.
또한, PGPR 균주는 식물성 호르몬의 일종인 IAA(indole-3-acetic acid) 등을 생성하여 직접적으로 식물 성장에 도움을 주며, 간접적으로는 항생물질, 용균효소, 전신유도내성 등을 통해 식물이 성장하는데 도움을 주는 것으로 알려져 있다.In addition, PGPR strains directly help plant growth by producing IAA (indole-3-acetic acid), a kind of plant hormone, and indirectly, plant growth through antibiotics, lytic enzymes, systemic induced tolerance, etc. It is known to help with
식물성 호르몬인 에틸렌(ethylene)은 농업 품질관리에 중요한 역할을 해 왔으며, 식물이 성장 및 발달을 하는 동안에는 에틸렌의 수준은 0.05 ㎕/L로 낮은 편이지만, 식물이 노화되는 과정 또는 열매가 익어가는 기간에는 100 ㎕/L 이상의 고농도 에틸렌이 합성된다. Ethylene, a plant hormone, has played an important role in agricultural quality control. During plant growth and development, the level of ethylene is as low as 0.05 μl/L, but during plant aging or fruit ripening period ethylene at a high concentration of 100 μl/L or more is synthesized.
적정량의 에틸렌은 식물 성장의 초기 단계에서 발아와 성장에 도움을 주지만, 고농도의 에틸렌은 뿌리 신장의 저해를 일으키는데, 극심한 온도 변화, 수분, 자외선, 질병과 같은 환경 스트레스에 빈번하게 노출되면 에틸렌의 농도가 증가되며, 노화와 세포 손상이 가속화된다. Appropriate amounts of ethylene help germination and growth in the early stages of plant growth, but high concentrations of ethylene cause inhibition of root elongation. is increased, and aging and cell damage are accelerated.
PGPR 균주에 의해 생성되는 ACC(1-aminocyclopropane-1-carboxylate) 탈아미노효소(deaminase)는 에틸렌의 전구체인 ACC를 암모니아와 알파-케토부티레이트(α-ketobutyrate)로 분해하여 에틸렌의 농도를 낮춤으로써 환경 스트레스를 완화시켜 식물의 성장 촉진에 도움을 준다.ACC (1-aminocyclopropane-1-carboxylate) deaminase, produced by PGPR strains, decomposes ACC, a precursor of ethylene, into ammonia and α-ketobutyrate, thereby lowering the concentration of ethylene. It relieves stress and helps promote plant growth.
엑소 엔자임(exoenzyme) 또는 세포외 효소는 세포에서 분비되고 해당 세포 외부에서 기능하는 효소이다. 아밀라아제(amylase), 프로테아제(protease), 셀룰라아제(cellulase), 뉴클레아제(nuclease), 리파아제(lipase), 포스파타아제(phosphatase) 등의 가수분해 효소 등이 이에 포함된다. 엑소 엔자임은 원핵 세포와 진핵 세포 모두에 의해 생성되며 많은 생물학적 과정에서 중요한 구성 요소인 것으로 나타났다. 대부분의 경우 이러한 효소는 더 큰 거대 분자의 분해에 관여한다. 이러한 큰 거대 분자의 분해는 구성 성분이 세포막을 통과하여 세포로 들어가는 데 중요하다. 엑소 엔자임 활동에 의해 생성된 소분자는 세포로 들어가 다양한 세포 기능에 활용된다. An exoenzyme or extracellular enzyme is an enzyme that is secreted by a cell and functions outside that cell. Hydrolytic enzymes such as amylase, protease, cellulase, nuclease, lipase, and phosphatase are included therein. Exoenzymes are produced by both prokaryotic and eukaryotic cells and have been shown to be important components in many biological processes. In most cases, these enzymes are involved in the breakdown of larger macromolecules. The breakdown of these large macromolecules is important for their constituents to cross the cell membrane and enter the cell. Small molecules produced by exoenzyme activity enter cells and are utilized for various cellular functions.
또한, 박테리아와 균류는 그들의 환경에서 영양소를 소화하기 위해 엑소 엔자임을 생산하거나, 식물 병원성 곰팡이의 세포벽을 분해시키는 용균작용(degradative parasitism)을 수행해 균주가 식물병 방제 활성을 갖도록 하기도 한다. 이러한 유기체는 그러한 엑소 엔자임의 존재와 기능을 확인하기 위해 실험실 분석을 수행하는 데 사용될 수 있다. In addition, bacteria and fungi produce exoenzymes to digest nutrients in their environment, or perform degradative parasitism to degrade the cell walls of plant pathogenic fungi, so that strains have plant disease control activity. These organisms can be used to perform laboratory assays to confirm the presence and function of such exoenzymes.
미생물 엑소 엔자임이 제공하는 또 다른 중요한 역할은 육상 및 해양 환경의 자연 생태학과 생물학적 정화에 있다.Another important role served by microbial exoenzymes is in the natural ecology and bioremediation of terrestrial and marine environments.
본 발명은 식물 생장 촉진 및 식물병 방제 활성을 갖는 마이크로박테리움 테스타세움(Microbacterium testaceum) DDP398 균주에 관한 것으로, 식물의 근권(rhizosphere)으로부터 질소고정능, IAA 생성능, 사이드로포어 생성능, ACC 탈아미노효소 활성 및 세포외 효소 활성이 우수하며, 특히 식물 병원성 곰팡이 5종에 대한 항진균 효과를 보이고, 또한 다양한 미네랄 가용화능을 갖고 있다. 상기 식물 생장 촉진 및 식물병 방제 활성을 갖는 균주를 분리하고, 마이크로박테리움 테스타세움(Microbacterium testaceum) DDP398로 명명하였다.The present invention relates to a Microbacterium testaceum DDP398 strain having plant growth promotion and plant disease control activities, nitrogen fixation ability, IAA production ability, siderophore production ability, ACC desorption from the rhizosphere of plants. It has excellent amino enzyme activity and extracellular enzyme activity, shows antifungal effect against 5 kinds of plant pathogenic fungi, and also has various mineral solubilizing ability. The strain having the plant growth promoting and plant disease controlling activity was isolated and named as Microbacterium testaceum DDP398.
마이크로박테리움 테스타세움(Microbacterium testaceum) DDP398 균주의 식물 생장 촉진 및 식물병 방제 용도를 기술적 특징으로 선행문헌을 검색한 결과, 같은 속(genus) 또는 종(species)이거나 같은 용도의 하기와 같은 내용들이 개시되어 있다.Microbacterium testaceum ( Microbacterium testaceum ) As a result of searching prior literature for plant growth promotion and plant disease control use of DDP398 strain with technical characteristics, the following contents of the same genus or species or the same use are disclosed.
우선 첫째로, 본 발명의 균주와 동일한 종에 속하는 마이크로박테리움 테스타세움(Microbacterium testaceum) 균주(NRRL 기탁 No.X19)의 식물 생장 촉진(인산염 가용화능, 사이드로포어 생성능) 항진균 활성 및 세포외 효소(cellulase, chitinase) 활성을 통하여 식물 생장 촉진 용도 및 식물병 방제 용도를 개시하고 있고, 둘째로 본 발명의 균주와 동일한 종에 속하는 마이크로박테리움 테스타세움(Microbacterium testaceum) 균주(LAP2-26)의 식물 생장 촉진(질소고정) 용도를 개시하고 있고, 셋째로 본 발명의 균주와 동일한 속에 속하는 마이크로박테리움 아우룸(Microbacterium aurum) MA-8 균주의 상추균핵병균(Sclerotinia sclerotiorum)에 대한 항진균 활성을 통하여 식물병 방제 용도를 개시하고 있다.First of all, the plant growth promotion (phosphate solubilization ability, siderophore production ability) antifungal activity and extracellular Plant growth promotion use and plant disease control use are disclosed through enzyme (cellulase, chitinase) activity, and secondly, Microbacterium testaceum belonging to the same species as the strain of the present invention ( Microbacterium testaceum ) strain (LAP2-26) It discloses the plant growth promotion (nitrogen fixation) use of, and thirdly, the antifungal activity of the Microbacterium aurum MA-8 strain belonging to the same genus as the strain of the present invention against Sclerotinia sclerotiorum It discloses the use of plant disease control through.
상기 선행문헌들에서는 식물 생장 촉진 및 식물병 방제 용도라는 본 발명과 같은 용도를 개시하고 있지만, 이는 본 발명의 균주와 동속 또는 동종의 균주에 관한 것으로 본 발명의 마이크로박테리움 테스타세움 DDP398 균주와 동일한 균주의 식물 생장 촉진 및 식물 방제 활성 용도를 개시한 선행문헌은 없었다.Although the above prior literature discloses the same use as the present invention, which is for plant growth promotion and plant disease control, it relates to the strain of the same or the same species as the strain of the present invention, and the Microbacterium testaceum DDP398 strain of the present invention There is no prior literature disclosing the use of the same strain for plant growth promotion and plant control activity.
따라서, 신규한 마이크로박테리움 테스타세움 DDP398 균주의 식물 생장 촉진 및 식물병 방제능을 새로운 균주를 스크리닝하여 본발명을 완성하였다.Therefore, the present invention was completed by screening new strains for plant growth promotion and plant disease control of the novel Microbacterium testaceum DDP398 strain.
본 발명의 목적은 식물 생장 촉진 및 식물병 방제 활성을 갖는 마이크로박테리움 테스타세움(Microbacterium testaceum) DDP398 균주 및 이의 용도를 제공하는 것이다.An object of the present invention is to provide a Microbacterium testaceum DDP398 strain having plant growth promoting and plant disease control activities and uses thereof.
상기 목적을 달성하기 위하여, 본 발명은 수탁번호 KFCC 11880P로 기탁된 마이크로박테리움 테스타세움(Microbacterium testaceum) DDP398 균주를 제공한다.In order to achieve the above object, the present invention provides a Microbacterium testaceum DDP398 strain deposited under accession number KFCC 11880P.
또한, 본 발명은 상기 균주, 이의 배양물 또는 이들의 추출물을 유효성분으로 함유하는 식물 생장 촉진용 조성물을 제공한다.In addition, the present invention provides a composition for promoting plant growth containing the strain, its culture or extract thereof as an active ingredient.
또한, 본 발명은 상기 균주, 이의 배양물 또는 이들의 추출물을 유효성분으로 함유하는 식물병 방제용 조성물을 제공한다.In addition, the present invention provides a composition for controlling plant diseases containing the strain, its culture or extract thereof as an active ingredient.
상기 추출물은 상기 균주 및/또는 이의 배양물을 포함한다.The extract includes the strain and/or its culture.
또한, 본 발명은 상기 식물 생장 촉진용 조성물을 식물, 이의 종자 또는 이의 서식지에 처리하는 단계를 포함하는 식물 생장 촉진 방법을 제공한다.In addition, the present invention provides a plant growth promotion method comprising the step of treating a plant, its seed or its habitat with the composition for promoting plant growth.
또한, 본 발명은 상기 식물병 방제용 조성물을 식물, 이의 종자 또는 이의 서식지에 처리하는 단계를 포함하는 식물병 방제 방법을 제공한다.In addition, the present invention provides a plant disease control method comprising the step of treating a plant, its seed or its habitat with the composition for controlling plant disease.
본 발명의 마이크로박테리움 테스타세움(Microbacterium testaceum) DDP398 균주는 인산 가용화능, 인산아연 가용화능, 탄산칼슘 가용화능, 규산마그네슘 가용화능, 질소 고정화능, IAA 생성능, 사이드로포어 생성능, ACC 탈아미노효소 활성 및 세포외 효소 활성을 가지므로, 상기 균주, 이의 배양물 또는 이들의 추출물을 유효성분으로 함유하는 조성물은 식물 생장 촉진 및 식물병 방제에 유용하게 사용될 수 있다. Microbacterium testaceum DDP398 strain of the present invention has phosphoric acid solubilization ability, zinc phosphate solubilization ability, calcium carbonate solubilization ability, magnesium silicate solubilization ability, nitrogen fixation ability, IAA production ability, siderophore production ability, ACC deamination Since it has enzymatic activity and extracellular enzyme activity, a composition containing the strain, its culture or its extract as an active ingredient can be usefully used for plant growth promotion and plant disease control.
도 1a는 DDP77와 DDP398 균주의 IAA(indole-3-acetic acid) 생성능을 측정한 결과 그래프이다.
도 1b는 DDP77와 DDP398 균주의 시데로포어(siderophore) 생성능을 평가한 사진이다.
도 2a는 DDP77와 DDP398 균주의 인산가용화능을 평가한 사진이다.
도 2b는 DDP77와 DDP398 균주의 ZnO, Zn3(PO4)2·4H2O, ZnCO3 가용화능을 평가한 사진이다.
도 2c는 DDP77와 DDP398 균주의 탄산칼슘 가용화능을 평가한 사진이다.
도 2d는 DDP77와 DDP398 균주의 규소 가용화능을 평가한 사진이다.
도 3은 마이크로박테리움 테스타세움(Microbacterium testaceum) DDP398 균주의 16s rRNA 염기서열 분석을 통해 작성한 계통도이다.
도 4는 마이크로박테리움 테스타세움(Microbacterium testaceum) DDP398 균주의 주사전자 현미경(Scanning electron microscope, SEM) 사진이다.1a is a graph showing the results of measuring indole-3-acetic acid (IAA) producing ability of DDP77 and DDP398 strains.
Figure 1b is a photograph evaluating the siderophore production ability of DDP77 and DDP398 strains.
Figure 2a is a photograph evaluating the phosphoric acid solubilization ability of DDP77 and DDP398 strains.
Figure 2b is a photograph of the evaluation of ZnO, Zn 3 (PO 4 ) 2 4H 2 O , ZnCO 3 solubilizing ability of DDP77 and DDP398 strains.
Figure 2c is a photograph evaluating the calcium carbonate solubilization ability of DDP77 and DDP398 strains.
Figure 2d is a photograph evaluating the silicon solubilization ability of DDP77 and DDP398 strains.
3 is a phylogenetic diagram created through 16s rRNA sequencing analysis of Microbacterium testaceum DDP398 strain.
4 is a scanning electron microscope (SEM) picture of Microbacterium testaceum DDP398 strain.
이하 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
마이크로박테리움 테스타세움의 분류는 다음과 같다:The classification of Microbacterium testaceum is as follows:
역: 박테리아(Bacteria), Inverse: Bacteria,
문: 방선균(Actinobacteria),Phylum: Actinobacteria,
강: 방선균(Actinomycetia),Class: Actinomycetia,
목: 마이크로코칼레스(Micrococcales),Order: Micrococcales,
과: 마이크로박테리아세아에(Microbacteriaceae)Family: Microbacteriaceae
속: 마이크로박테리움(Microbacterium), Genus: Microbacterium,
종: 테스타세움(Testaceum) Species: Testaceum
마이크로박테리움(Microbacterium) 속은 높은 GC 함량 방선균(Actinobacteria) 강(class)에 속한다. 마이크로박테리움(Microbacterium) 속의 구성원은 토양, 곤충, 인간 임상 표본, 해양 환경, 식물, 유제품 등을 포함한 광범위한 환경에서 분리될 수 있다.The genus Microbacterium belongs to the class Actinobacteria with high GC content. Members of the genus Microbacterium can be isolated from a wide range of environments, including soil, insects, human clinical specimens, marine environments, plants, dairy products, and the like.
마이크로박테리움 테스타세움(Microbacterium testaceum)은 질병 증상을 일으키지 않고 식물 숙주 내에 거주하는 내생 세균이다. Microbacterium testaceum is an endogenous bacterium that resides within plant hosts without causing disease symptoms.
본 발명은 수탁번호 KFCC 11880P로 기탁된 마이크로박테리움 테스타세움(Microbacterium testaceum) DDP398 균주를 제공한다.The present invention provides a Microbacterium testaceum DDP398 strain deposited under accession number KFCC 11880P.
상기 균주는 서열번호 1의 16S rRNA 서열을 가진다.The strain has the 16S rRNA sequence of SEQ ID NO: 1.
상기 균주는 토양 또는 식물 뿌리로부터 분리된 것이다.The strain is isolated from soil or plant roots.
상기 균주는 식물 생장 촉진 활성을 가지고, 식물병 방제 활성을 가진다.The strain has plant growth promoting activity and has plant disease control activity.
상기 균주의 식물 생장 촉진 활성은 인산 가용화능, 인산아연 가용화능, 탄산칼슘 가용화능, 규산마그네슘 가용화능, 질소 고정화능, IAA 생성능, 사이드로포어 생성능, ACC 탈아미노효소 활성 및 세포외 효소 활성으로 구성되는 군으로부터 선택되는 어느 하나 이상의 활성에 의한 것이다.The plant growth promoting activity of the strain is phosphate solubilization ability, zinc phosphate solubilization ability, calcium carbonate solubilization ability, magnesium silicate solubilization ability, nitrogen fixation ability, IAA production ability, siderophore production ability, ACC deaminase activity and extracellular enzyme activity. It is due to any one or more activities selected from the group consisting of
상기 균주의 식물병 방제 활성은 사이드로포어 생성능에 의한 것이다.The plant disease control activity of the strain is due to the ability to produce siderophores.
상기 균주의 식물병 방제 활성은 세포외 효소 활성에 의한 것이다.Plant disease control activity of the strain is due to extracellular enzyme activity.
상기 균주는 10 내지 60℃, 바람직하게는 10 내지 50℃, 보다 바람직하게는 10 내지 35℃에서 생장할 수 있고, pH 5 내지 11에서 생장할 수 있으며, 염(NaCl) 농도가 0 내지 10%인 배지에서 생장할 수 있다.The strain can grow at 10 to 60 ° C, preferably 10 to 50 ° C, more preferably 10 to 35 ° C, grow at pH 5 to 11, and have a salt (NaCl) concentration of 0 to 10%. Can grow on phosphorus medium.
또한, 본 발명은 수탁번호 KFCC 11880P로 기탁된 마이크로박테리움 테스타세움(Microbacterium testaceum) DDP398, 이의 배양물 또는 이들의 추출물을 유효성분으로 함유하는 식물 생장 촉진용 조성물을 제공한다.In addition, the present invention provides a composition for promoting plant growth containing, as an active ingredient, Microbacterium testaceum DDP398, a culture thereof, or an extract thereof deposited under accession number KFCC 11880P.
상기 마이크로박테리움 테스타세움(Microbacterium testaceum) DDP398 균주는 상술한 바와 같은 특징을 가진다. The Microbacterium testaceum ( Microbacterium testaceum ) DDP398 strain has the characteristics as described above.
일례로, 상기 균주는 서열번호 1의 16S rRNA 서열을 가진다.As an example, the strain has a 16S rRNA sequence of SEQ ID NO: 1.
또한, 상기 균주는 토양 또는 식물 뿌리로부터 분리된 것이다.In addition, the strain is isolated from soil or plant roots.
또한, 상기 균주는 식물 생장 촉진 활성을 가지고, 식물병 방제 활성을 가진다In addition, the strain has a plant growth promoting activity and a plant disease control activity.
상기 배양물은 상기 마이크로박테리움 테스타세움(Microbacterium testaceum) DDP398 균주를 배양한 배양액 또는 이를 물리적 방법으로 여과한 것이다.The culture is a culture medium in which the Microbacterium testaceum DDP398 strain is cultured or filtered by a physical method.
상기 조성물은 상기 균주, 이의 배양물 또는 이들의 추출물에 불활성 담체를 혼합하고, 상기 혼합물이 유제, 유액, 유동화제, 습윤성 분말, 과립화 습윤성 분말, 분말제, 과립제 등으로 제형화 될 수 있도록 혼합물에 계면활성제 및 필요한 기타 보조제를 첨가함으로써 제조된다.The composition is a mixture by mixing an inert carrier with the strain, its culture or extract thereof, and formulating the mixture into an emulsion, emulsion, glidant, wettable powder, granulated wettable powder, powder, granule, etc. It is prepared by adding a surfactant and other necessary auxiliary agents to.
제형에서 사용될 수 있는 액체 담체의 예는 물; 알콜, 예로 메탄올 및 에탄올; 케톤, 예로 아세톤 및 메틸 에틸 케톤; 방향족 탄화수소, 예로 벤젠, 톨루엔, 자일렌, 에틸벤젠 및 메틸나프탈렌; 지방족 탄화수소, 예로 헥산, 시클로헥산, 케로신 및 라이트 오일; 에스테르, 예로 에틸 아세테이트 및 부틸 아세테이트; 니트릴, 예로 아세토니트릴 및 이소부티르니트릴; 에테르, 예로 디이소프로필에테르 및 디옥산; 산 아미드, 예로 N,N-디메틸 포름아미드 및 N,N-디메틸아세트아미드; 할로겐화 탄화수소, 예로 디클로로메탄, 트리클로로에탄 및 사염화탄소; 디메틸 술폭시드; 및 식물성 오일, 예로 대두유 및 면실유가 포함된다.Examples of liquid carriers that may be used in the formulation include water; alcohols such as methanol and ethanol; ketones such as acetone and methyl ethyl ketone; aromatic hydrocarbons such as benzene, toluene, xylene, ethylbenzene and methylnaphthalene; aliphatic hydrocarbons such as hexane, cyclohexane, kerosene and light oil; esters such as ethyl acetate and butyl acetate; nitriles such as acetonitrile and isobutyrnitrile; ethers such as diisopropylether and dioxane; acid amides such as N,N-dimethyl formamide and N,N-dimethylacetamide; halogenated hydrocarbons such as dichloromethane, trichloroethane and carbon tetrachloride; dimethyl sulfoxide; and vegetable oils such as soybean oil and cottonseed oil.
제형에서 사용될 수 있는 고체 담체의 예는 미세 분말 또는 과립 예컨대 광물 예컨대 카올린 점토, 애터펄자이트 점토, 벤토나이트, 몬트모릴로나이트, 애시드 화이트 점토, 피로필라이트, 탈크, 규조토 및 탈사이트; 천연 유기 물질 예컨대 옥수수 잎대 분말 및 월넛 껍질 분말; 합성 유기 물질 예컨대 우레아; 염 예컨대 탄산 칼슘 및 황산 암모늄; 합성 무기 물질 예컨대 합성 수화 산화 규소를 포함하며; 액체 담체로서, 방향족 탄화수소 예컨대 자일렌, 알킬벤젠 및 메틸나프탈렌; 알코올 예컨대 2-프로판올, 에틸렌글리콜, 프로필렌 글리콜 및 에틸렌글리콜 모노에틸 에테르; 케톤 예컨대 아세톤, 시클로헥사논 및 이소포론; 식물성 오일 예컨대 대두유 및 면실유; 석유 지방족 탄화수소, 에스테르, 디메틸술폭시드, 아세토니트릴 및 물을 포함한다.Examples of solid carriers that can be used in the formulation include fine powders or granules such as minerals such as kaolin clay, attapulgite clay, bentonite, montmorillonite, acid white clay, pyrophyllite, talc, diatomaceous earth and talcite; natural organic substances such as corn stalk meal and walnut husk meal; synthetic organic substances such as urea; salts such as calcium carbonate and ammonium sulfate; synthetic inorganic materials such as synthetic hydrated silicon oxide; As liquid carriers, aromatic hydrocarbons such as xylenes, alkylbenzenes and methylnaphthalenes; alcohols such as 2-propanol, ethylene glycol, propylene glycol and ethylene glycol monoethyl ether; ketones such as acetone, cyclohexanone and isophorone; vegetable oils such as soybean oil and cottonseed oil; petroleum aliphatic hydrocarbons, esters, dimethylsulfoxide, acetonitrile and water.
계면활성제의 예는 음이온성 계면활성제 예컨대 알킬 술페이트 에스테르 염, 알킬아릴 술포네이트 염, 디알킬술포숙시네이트 염, 폴리옥시에틸렌 알킬아릴 에테르 포스페이트 에스테르 염, 리그노술포네이트 염 및 나프탈렌술포네이트 포름알데히드 중축합물; 및 비이온성 계면활성제 예컨대 폴리옥시에틸렌 알킬 아릴 에테르, 폴리옥시에틸렌 알킬폴리옥시프로필렌 블럭 공중합체 및 소르비탄 지방산 에스테르 및 양이온성 계면활성제 예컨대 알킬트리메틸암모늄 염을 포함한다.Examples of surfactants include anionic surfactants such as alkyl sulfate ester salts, alkylaryl sulfonate salts, dialkylsulfosuccinate salts, polyoxyethylene alkylaryl ether phosphate ester salts, lignosulfonate salts and naphthalenesulfonate form aldehyde polycondensates; and nonionic surfactants such as polyoxyethylene alkyl aryl ethers, polyoxyethylene alkylpolyoxypropylene block copolymers and sorbitan fatty acid esters, and cationic surfactants such as alkyltrimethylammonium salts.
다른 제형 보조제의 예는 수용성 중합체 예컨대 폴리비닐 알코올 및 폴리비닐피롤리돈, 다당류 예컨대 아라비아고무, 알긴산 및 이의 염, CMC(카르복시메틸-셀룰로오스), 잔탄 고무, 무기 물질 예컨대 알루미늄 마그네슘 실리케이트 및 알루미나 졸(alumina sol), 보존제, 착색제 및 안정화제 예컨대 PAP(산 포스페이트 이소프로필) 및 BHT(부틸하이드록시톨루엔)를 포함한다.Examples of other formulation auxiliaries are water-soluble polymers such as polyvinyl alcohol and polyvinylpyrrolidone, polysaccharides such as gum arabic, alginic acid and its salts, CMC (carboxymethyl-cellulose), xanthan gum, inorganic materials such as aluminum magnesium silicate and alumina sol ( alumina sol), preservatives, colorants and stabilizers such as PAP (acid phosphate isopropyl) and BHT (butylhydroxytoluene).
또한, 본 발명은 수탁번호 KFCC 11880P로 기탁된 마이크로박테리움 테스타세움(Microbacterium testaceum) DDP398 균주, 이의 배양물 또는 이들의 추출물을 유효성분으로 함유하는 식물병 방제용 조성물을 제공한다.In addition, the present invention provides a composition for controlling plant diseases containing, as an active ingredient, the Microbacterium testaceum DDP398 strain deposited under accession number KFCC 11880P, a culture thereof, or an extract thereof.
또한, 본 발명은 상기 식물 생장 촉진용 조성물을 식물, 이의 종자 또는 이의 서식지에 처리하는 단계를 포함하는 식물 생장 촉진 방법을 제공한다.In addition, the present invention provides a plant growth promotion method comprising the step of treating a plant, its seed or its habitat with the composition for promoting plant growth.
상기 식물 생장 촉진용 조성물은 상술한 바와 같은 특징을 가진다. The composition for promoting plant growth has the same characteristics as described above.
일례로, 상기 조성물은 수탁번호 KFCC 11880P로 기탁된 마이크로박테리움 테스타세움(Microbacterium testaceum) DDP398 균주, 이의 배양물 또는 이들의 추출물을 유효성분으로 함유하는 조성물이다.As an example, the composition is a composition containing a Microbacterium testaceum DDP398 strain, a culture thereof, or an extract thereof deposited under accession number KFCC 11880P as an active ingredient.
상기 처리는 조성물을 식물체에 직접 살포하거나, 식물체가 자라고 있는 토양에 살포하거나, 식물체의 배양용 매개체에 살포하는 것이다.The treatment is to spray the composition directly on the plant, spray on the soil in which the plant is growing, or spray on the medium for culturing the plant.
상기 조성물을 식물체에 직접 살포하는 방법으로 예를 들어 줄기 및 잎에 분무하는 것과 같은 식물 표면 상의 적용이 포함된다.Methods of spraying the composition directly on plants include application on plant surfaces, such as spraying stems and leaves, for example.
상기 조성물을 식물체가 자라고 있는 토양에 살포하는 방법으로 예를 들어, 토양 상 분무, 토양과의 혼합, 액체 처리제의 토양 내로의 살포(액체 처리제의 관개(irrigation), 토양 내로의 주입, 액체 처리제의 적하)가 포함될 수 있으며, 처리되는 장소의 예는 재식혈(planting hole), 고랑, 재식혈 주변, 심을골(planting furrow) 주변, 성장 부위의 전체 표면, 토양과 식물 사이 부분, 뿌리 사이 부위, 식물체의 줄기 밑 부위, 주 고랑, 성장 토양, 못자리, 모 재배용 상자, 모 재배용 트레이, 모판을 포함한다.A method of spraying the composition to the soil in which plants are growing, for example, spraying on soil, mixing with soil, spraying a liquid treatment agent into the soil (irrigation of a liquid treatment agent, injection into the soil, liquid treatment agent dripping), examples of places to be treated include planting holes, furrows, around planting holes, around planting furrows, the entire surface of growing areas, areas between soil and plants, areas between roots, It includes the lower part of the stem of the plant, the main furrow, the growing soil, the seed bed, the seedling box, the seedling tray, and the seedling bed.
또한, 본 발명은 상기 식물병 방제용 조성물을 식물, 이의 종자 또는 이의 서식지에 처리하는 단계를 포함하는 식물병 방제 방법을 제공한다.In addition, the present invention provides a plant disease control method comprising the step of treating a plant, its seed or its habitat with the composition for controlling plant disease.
본 발명의 구체적인 실시예에서, 본 발명자들은 대한민국 10개 지점의 토양 및 식물 뿌리로부터 균주를 분리하고, 분리한 균주들에 대하여 인산 가용화능, 인산아연 가용화능, 탄산칼슘 가용화능, 규산마그네슘 가용화능, 질소 고정화능, IAA 생성능, 사이드로포어 생성능, ACC 탈아미노효소 활성 및 세포외 효소 활성을 평가하여 2종의 균주를 선별하였다.In a specific embodiment of the present invention, the present inventors isolated strains from soil and plant roots of 10 points in Korea, and tested phosphoric acid solubilization ability, zinc phosphate solubilization ability, calcium carbonate solubilization ability, and magnesium silicate solubilization ability for the isolated strains. , Nitrogen fixation ability, IAA production ability, siderophore production ability, ACC deaminase activity and extracellular enzyme activity were evaluated to select two strains.
마이크로박테리움 테스타세움(Microbacterium testaceum) DDP77 및 DDP398 균주 모두 중간 정도의 질소고정화능, IAA 생성능, 사이드로포어 생성능 및 ACC 탈아미노효소 활성을 나타냈다(표 2). Microbacterium testaceum ( Microbacterium testaceum ) Both DDP77 and DDP398 strains showed moderate nitrogen fixation ability, IAA production ability, siderophore production ability and ACC deaminase activity (Table 2).
또한 미네랄 가용화능에 있어서는 중간 정도의 인산아연, 탄산칼슘 및 규산마그네슘 가용화능을 보였고, 특히 DDP77 균주는 중간 정도의 인산가용화능을 보인 반면, DDP398 균주는 강한 인산가용화능을 보였다(표 3). In addition, in terms of mineral solubilization ability, zinc phosphate, calcium carbonate, and magnesium silicate solubilization ability were moderate, and in particular, strain DDP77 showed moderate phosphate solubilization ability, whereas strain DDP398 showed strong phosphate solubilization ability (Table 3).
뿐만 아니라, 3종의 세포외 효소 활성이 비슷한 수준을 나타냈고, 프로테아제는 DDP398이 0.85mg/ml의 농도로 DDP77의 0.58mg/ml 대비 68% 높게 나타났다(표 4).In addition, the three types of extracellular enzyme activity showed similar levels, and the protease showed a concentration of 0.85 mg / ml for DDP398, which was 68% higher than that of 0.58 mg / ml for DDP77 (Table 4).
이들의 생장조건을 확인하여 10 내지 35℃ 넓은 범위의 온도,5 내지 11의 pH 및 0내지 10%의 염 농도 조건에서 생장할 수 있으며, 다양한 식물 생장 촉진 활성을 가지는 DDP398 균주를 최종적으로 선별하였다.After confirming their growth conditions, DDP398 strains, which can grow in a wide range of temperature from 10 to 35 ° C, pH from 5 to 11 and salt concentration from 0 to 10%, and have various plant growth promoting activities, were finally selected. .
또한, 본 발명자들은 선별된 DDP398 균주를 동정하여 마이크로박테리움 테스타세움(Microbacterium testaceum) DDP398 균주로 명명하고 한국미생물보존센터에 수탁번호 KFCC 11880P로 기탁하였으며, 상기 균주의 형태학적 및 생리학적 특성을 확인하였다In addition, the present inventors identified the selected DDP398 strain, named it Microbacterium testaceum DDP398 strain, and deposited it with the Korea Microorganism Conservation Center under the accession number KFCC 11880P, and the morphological and physiological characteristics of the strain confirmed
DDP398 균주는 막대 모양인 간균(bacillus)의 형태를 가짐을 확인하였다(도 4).It was confirmed that the DDP398 strain had the form of rod-shaped bacillus (FIG. 4).
또한, Alkaline phosphatase, Esterase(C4), Esterase(C8), Leucine arylamidase, Valine arylamidase, Crystine arylamidase, α-chymotrypsin, Acid phosphatase, Naphtol-AS-BI-phosphohydrolase, α-glucosidase, β-glucuronidase, α-glucosidase 및 β-glucosidase 효소 활성을 나타났으며, 그 중에서도 Leucine arylamidase와 α-glucosidase에서 강한 효소 활성을 나타내었다(표 7).In addition, alkaline phosphatase, esterase (C4), esterase (C8), leucine arylamidase, valine arylamidase, crystaline arylamidase, α-chymotrypsin, acid phosphatase, naphtol-AS-BI-phosphohydrolase, α-glucosidase, β-glucuronidase, α-glucosidase and β-glucosidase enzyme activities, among which strong enzyme activities were shown in leucine arylamidase and α-glucosidase (Table 7).
따라서, 본 발명의 마이크로박테리움 테스타세움(Microbacterium testaceum) DDP398 균주(KFCC 11880P)는 인산 가용화능, 인산아연 가용화능, 탄산칼슘 가용화능, 규산마그네슘 가용화능, 질소 고정화능, IAA 생성능, 사이드로포어 생성능, ACC 탈아미노효소 활성 및 세포외 효소 활성을 가지므로, 식물 생장 촉진 및 식물병 방제에 유용하게 사용될 수 있다.Therefore, the Microbacterium testaceum DDP398 strain (KFCC 11880P) of the present invention has phosphoric acid solubilization ability, zinc phosphate solubilization ability, calcium carbonate solubilization ability, magnesium silicate solubilization ability, nitrogen fixing ability, IAA production ability, sideways Since it has pore-forming ability, ACC deaminase activity and extracellular enzyme activity, it can be usefully used for plant growth promotion and plant disease control.
이하 본 발명을 실시예에 의해 상세히 설명한다.Hereinafter, the present invention will be described in detail by examples.
단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 의해서 한정되는 것은 아니다.However, the following examples are only to illustrate the present invention, and the content of the present invention is not limited by the following examples.
토양으로부터 신규 균주들의 분리Isolation of novel strains from soil
근권(rhizosphere) 토양으로부터 신규 균주들을 분리하기 위하여, 삼락공원 갈대밭(부산광역시, 대한민국), 다대포해수욕장 갈대밭 및 해수욕장(부산광역시, 대한민국), 신라대학교 인근 야산(부산광역시, 대한민국), 대저 토마토 밭(부산광역시, 대한민국), 기장군 배 밭(부산광역시, 대한민국), 창원시 인근 야산 및 텃밭(경상남도, 대한민국), 제주시 노리매 공원 및 인근 해변(제주도, 대한민국)의 10개 지점으로부터 토양과 식물 뿌리들을 각각 채취하였다. In order to isolate new strains from rhizosphere soil, reed fields in Samnak Park (Busan Metropolitan City, South Korea), Dadaepo Beach reed fields and beaches (Busan Metropolitan City, South Korea), hills near Silla University (Busan Metropolitan City, South Korea), Daejeo Tomato Soil and plant roots from 10 points: a field (Busan Metropolitan City, South Korea), a pear field in Gijang-gun (Busan Metropolitan City, South Korea), a hill and vegetable garden near Changwon-si (Gyeongsangnam-do, South Korea), and Norimae Park and nearby beaches in Jeju-si (Jeju Island, South Korea) were each collected.
본 발명의 Microbacterium testaceum DDP77 균주는 부산시 다대포 해수욕장 갈대밭 토양에서 주로 분리되었고, Microbacterium testaceum DDP398 균주는 초음파 처리된 다대포 해수욕장 갈대밭 뿌리 시료에서 분리되었다. The Microbacterium testaceum DDP77 strain of the present invention was mainly isolated from the reed field soil of Dadaepo Beach, Busan, and the Microbacterium testaceum DDP398 strain was isolated from the ultrasonically treated Dadaepo Beach reed field root sample.
채집된 토양과 뿌리를 음지에서 2일간 풍건하였으며, 식물 뿌리들을 포함하는 토양 시료 각 1 g을 9 mL의 멸균된 생리식염수에 현탁하였다. 각 현탁액을 연속적으로 희석하여 각 25 개의 농도로 만든 후, 5 가지 평판 배지인 BA (Bennet agar, 1% glucose, 0.2% peptone, 0.1% beef extract, 0.1% yeast extract, 1.5% agar), GMSA (glucose minimal salts agar, 0.2% glucose, 1% yeast extract, 0.2% KH2PO4, 0.3% K2HPO4, 0.01% MgSO4·7H2O, 0.0002% MnSO4, 1.5% agar), PDA (potato dextrose agar, DifcoTM, 0.4% potato starch, 2% dextrose, 1.5% agar), TSA (tryptic soy agar, DifcoTM , 1.7% pancreatic digest of casein, 0.3% papaic digest of soybean, 0.25% dextrose, 0.5% NaCl, 0.25% K2HPO4, 1.5% agar) 및 HV (humic-vitamin agar, 0.1% humic acid, 0.05% KCl, 0.05% Na2HPO4, 0.05% MgSO4, 0.002% CaCO3, 0.002% FeSO4, 0.1% VB stock solution, 1.5% agar)에 0.1 mL씩 도말하여 30℃에서 24시간 동안 배양하였다. The collected soil and roots were air-dried in the shade for 2 days, and each 1 g soil sample containing plant roots was suspended in 9 mL of sterilized physiological saline. After serially diluting each suspension to make 25 concentrations, 5 types of plate medium BA (Bennet agar, 1% glucose, 0.2% peptone, 0.1% beef extract, 0.1% yeast extract, 1.5% agar), GMSA ( glucose minimal salts agar, 0.2% glucose, 1% yeast extract, 0.2% KH 2 PO 4 , 0.3% K 2 HPO 4 , 0.01% MgSO 4 7H 2 O, 0.0002% MnSO 4 , 1.5% agar), PDA (potato dextrose agar, Difco TM , 0.4% potato starch, 2% dextrose, 1.5% agar), TSA (tryptic soy agar, Difco TM , 1.7% pancreatic digest of casein, 0.3% papaic digest of soybean, 0.25% dextrose, 0.5% NaCl , 0.25% K 2 HPO 4 , 1.5% agar) and HV (humic-vitamin agar, 0.1% humic acid, 0.05% KCl, 0.05% Na 2 HPO 4 , 0.05% MgSO 4 , 0.002% CaCO 3 , 0.002% FeSO 4 , 0.1% VB stock solution, 1.5% agar) were smeared in 0.1 mL each and incubated at 30 ° C for 24 hours.
채집한 식물 뿌리로부터 내생진균류를 분리하기 위하여, 각 식물 뿌리 시료를 계면활성제(Tween 80)와 표백제 (perchloric acid 1.0%)로 살균처리하고 멸균수로 세척한 후 초음파처리(sonication)하였다. 초음파처리된 뿌리 시료를 상기 각 5가지 평판 배지에 올려서 30℃에서 24시간 동안 배양하였다. 미생물이 자란 배지 상에서 형태나 색깔이 다른 콜로니(colony)를 계대 배양하여 510종 균주를 분리하였다.In order to separate endophytic fungi from the collected plant roots, each plant root sample was sterilized with a surfactant (Tween 80) and bleach (perchloric acid 1.0%), washed with sterile water, and then subjected to sonication. The sonicated root samples were placed on each of the five plate media and cultured at 30° C. for 24 hours. 510 strains were isolated by subculturing colonies of different shapes and colors on a culture medium in which microorganisms were grown.
실험예 1. 분리한 균주의 식물 생장 촉진 활성Experimental Example 1. Plant growth promoting activity of the isolated strain
1-1. 분리한 균주의 질소 고정화능 확인1-1. Confirmation of nitrogen fixation ability of the isolated strain
대기 중의 질소를 고정하여 생물체가 이용할 수 있는 질산염 형태로 질소원을 제공하면 식물 생장에 도움을 줄 수 있으므로, 분리 균주들의 질소 고정화능을 측정하였다.Fixing nitrogen in the atmosphere and providing a nitrogen source in the form of nitrate that can be used by organisms can help plant growth, so separation The nitrogen fixation capacity of the strains was measured.
구체적으로, 질소 고정세균 분리용 NFB(Nitrogen Free Bromothymol blue) [(HO2CCH2CH(OH)CO2H, 0.05% K2HPO4, 0.001% MgSO4·7H2O, 0.002% NaCl, 0.005% FeSO4·7H2O, 0.0002% Na2MoO4, 0.001% MnSO4·7H2O, 0.001% CaCl2, 0.4% KOH, 0.2% bromothymol blue (in 0.5% alcohol), 0.175% agar, pH 6.8] 배지를 이용하였다.Specifically, NFB (Nitrogen Free Bromothymol blue) for isolation of nitrogen-fixing bacteria [(HO 2 CCH 2 CH(OH)CO 2 H, 0.05% K 2 HPO 4 , 0.001% MgSO 4 7H 2 O, 0.002% NaCl, 0.005 % FeSO 4 7H 2 O, 0.0002% Na 2 MoO 4 , 0.001% MnSO 4 7H 2 O, 0.001% CaCl 2 , 0.4% KOH, 0.2% bromothymol blue (in 0.5% alcohol), 0.175% agar, pH 6.8 ] medium was used.
상기 배지는 무기염 및 탄소원만 함유하고 질소원은 결핍되어 있어 대기 중의 질소를 고정할 능력이 있는 균주만이 생장할 수 있는 선택배지이다. The medium contains only inorganic salts and a carbon source and lacks a nitrogen source, so that only strains capable of fixing nitrogen in the atmosphere can grow.
시험관에 NFB 배지 5 mL를 넣고 실시예 1에서 분리된 전배양 균주를 루프 (loop)로 접종하여 30℃에서 2일간 배양하면서 배지 색이 파란색으로 변할 경우 해당 균주가 대기 중의 질소를 고정하여 생장할 수 있는 균주인 것으로 판단하였다. Put 5 mL of NFB medium in a test tube, inoculate the precultured strain isolated in Example 1 with a loop, and incubate at 30 ° C for 2 days, when the color of the medium turns blue, the strain can grow by fixing nitrogen in the atmosphere. It was determined that it was a possible strain.
그 결과, DDP77 및 DDP398 균주에서 질소 고정화능이 있음을 확인하였다(표 1).As a result, it was confirmed that the DDP77 and DDP398 strains had nitrogen fixation ability (Table 1).
++: 강한 식물 생장 촉진 활성; +: 중간 정도의 식물 생장 촉진 활성++: strong plant growth promoting activity; +: Moderate plant growth promoting activity
1-2. 분리한 균주의 IAA 생성능 확인1-2. Confirmation of IAA-producing ability of the isolated strain
IAA(indole-3-acetic acid)는 식물생장 호르몬으로 식물의 신장을 촉진시키고 뿌리 신장 및 과실 형성 등을 촉진하므로, 분리 균주들의 IAA 생성능을 측정하였다.Since indole-3-acetic acid (IAA) is a plant growth hormone that promotes plant elongation and promotes root elongation and fruit formation, the ability of the isolated strains to produce IAA was measured.
구체적으로, 0.1%의 트립토판(tryptophan)을 첨가한 KB(King's B) 배지에 실시예 1에서 분리된 전배양 균주를 접종하여 30℃에서 130 rpm으로 2일간 배양하였다. 배양된 배양액을 원심분리(10,000 rpm, 15분)하여 상층액을 분리한 후, 살코프스키 시약(Salkowski's reagent; 35% HClO4 50 mL, 0.5 M FeCl3 1 mL)을 1:2 (v/v)의 비율로 첨가하고, 분홍색이 발색되는 동안 상온에서 30분간 정치하였다. 이후 흡광 광도계를 이용하여 530 nm에서 각 혼합액의 흡광도를 측정하였다. 표준물질로 IAA를 이용하여 상기와 같은 방법으로 실험한 후 검량선을 작성하여 상기 각 반응액 내의 IAA의 농도를 환산하였다.Specifically, the precultured strain isolated in Example 1 was inoculated into KB (King's B) medium supplemented with 0.1% tryptophan, and cultured at 30° C. at 130 rpm for 2 days. After separating the supernatant by centrifuging the culture medium (10,000 rpm, 15 minutes), Salkowski's reagent (35% HClO 4 50 mL, 0.5
그 결과, DDP77 및 DDP398 균주에서 IAA 생성능을 나타냄을 확인하였다(표 1 및 도 1a). As a result, it was confirmed that the DDP77 and DDP398 strains showed IAA producing ability (Table 1 and FIG. 1a).
1-3. 분리한 균주의 siderophore 생성능 확인1-3. Confirmation of the siderophore production ability of the isolated strain
사이드로포어는 철 이온에 특이적으로 결합하여 식물 생장에 직접적으로 도움을 주며, 식물병원균의 철 이온 흡수를 방해하여 생육을 억제하므로, CAS 블루 아가 방법(chrome azurol S blue agar plate assay)을 이용하여 분리 균주들의 사이드로포어 생성능을 측정하였다.Siderophores directly help plant growth by specifically binding to iron ions, and inhibit growth by interfering with the absorption of iron ions by plant pathogens. separated by The siderophore-producing ability of the strains was measured.
구체적으로, 60.5 mg의 CAS를 50 mL의 증류수에 녹이고, 10 mL의 Fe(Ⅲ) 용액(FeCl3·6H2O 27 mg, HCl 83.3 ㎕/100 mL 증류수)을 첨가하고, 72.9 mg의 HDTMA(hexa decyltrimethyl ammonium bromide)를 40 mL의 증류수에 녹인 후 첨가하여 CAS 지시용액을 준비하였다. 증류수 900 mL에 trytone 10 g, yeast extract 5 g, NaCl 5 g, agar 15 g을 넣어 배지를 만들고 CAS 지시용액과 섞은 후 pH 6.8로 맞춘 후 멸균하였다. 멸균된 CAS 블루 아가 플레이트 중앙을 코르크 보러를 이용하여 잘라낸 뒤, 분리된 균주의 상등액을 접종하여 30 ℃에서 4일간 배양시키면서 푸른색 배지에서 주황색 원(Orange halo)의 생성 유무를 관찰하여 사이드로포어 생성능을 측정하였다.Specifically, 60.5 mg of CAS was dissolved in 50 mL of distilled water, 10 mL of Fe(III) solution (FeCl 3 6H 2 O 27 mg, HCl 83.3 μl/100 mL distilled water) was added, and 72.9 mg of HDTMA ( hexa decyltrimethyl ammonium bromide) was dissolved in 40 mL of distilled water and then added to prepare a CAS indicator solution. A medium was prepared by adding 10 g of trytone, 5 g of yeast extract, 5 g of NaCl, and 15 g of agar to 900 mL of distilled water, mixed with the CAS indicator solution, adjusted to pH 6.8, and sterilized. After cutting the center of the sterilized CAS blue agar plate using a cork borer, inoculating the supernatant of the isolated strain and incubating it at 30 ° C for 4 days, observing the formation of an orange halo in the blue medium, the sideropore The production ability was measured.
그 결과, DDP77과 DDP398 균주에서 주황색 원(Orange halo)이 나타나 사이드로포어 생성능이 있음을 확인하였다(표 1 및 도 1b).As a result, an orange halo appeared in the DDP77 and DDP398 strains, confirming that they had the ability to generate sideropores (Table 1 and FIG. 1b).
1-4. 분리한 균주의 ACC deaminase 생성능 확인1-4. Confirmation of ACC deaminase production ability of the isolated strain
ACC 탈아미노효소는 식물 생장을 억제하는 에틸렌(ethylene)의 전구체인 ACC를 암모니아와 알파-케토부티레이트(α-ketobutyrate)로 분해함으로써 식물 생장에 도움을 줄 수 있으므로, 분리 균주의 ACC 탈아미노효소 활성을 측정하였다.ACC deaminase can help plant growth by decomposing ACC, a precursor of ethylene that inhibits plant growth, into ammonia and α-ketobutyrate. The ACC deaminase activity of the strain was measured.
구체적으로, 시험관에 5 mL의 TSB(tryptic soy broth) 배지를 넣고 실시예 1에서 분리한 전배양 균주를 각각 접종하여 30℃에서 130 rpm으로 48시간 동안 배양한 후, 원심분리하여 균체를 회수하였다. 회수한 균체는 DF(Dworkin-Foster) 염(salt) 배지(KH2PO4 0.4%, Na2HPO4 0.6%, MgSO4·7H2O 0.02%, FeSO4 0.0001%, H3BO3 0.000001%, MnSO4·H2O 0.00001%, ZnSO4 0.000007%, CuSO4 0.000005%, MoO3 0.000001%, 글루코스(glucose) 0.2%, C6H12O7 0.2%, C6H8O7 0.2%, pH 7.3)로 두 차례 세척하고 질소원으로 3 mM ACC가 첨가된 DF 염 배지에 접종하여 30℃에서 130 rpm으로 5일간 배양하였다. 대조구로는 질소원으로 아무것도 첨가하지 않은 DF 염 배지를 사용하였고, 600 nm에서 흡광도를 측정하여 ACC를 첨가한 배지에서 생장한 균체의 증가 정도를 확인하여 ACC 탈아미노효소 활성을 측정하였다.Specifically, 5 mL of TSB (tryptic soy broth) medium was placed in a test tube, and each of the precultured strains isolated in Example 1 was inoculated, cultured at 30 ° C. at 130 rpm for 48 hours, and then centrifuged to recover the cells. . The recovered cells were DF (Dworkin-Foster) salt medium (KH 2 PO 4 0.4%, Na 2 HPO 4 0.6%, MgSO 4 7H 2 O 0.02%, FeSO 4 0.0001%, H 3 BO 3 0.000001% , MnSO 4 H 2 O 0.00001%, ZnSO 4 0.000007%, CuSO 4 0.000005%, MoO 3 0.000001%, glucose 0.2%, C 6 H 12 O 7 0.2%, C 6 H 8 O 7 0.2%, pH 7.3), washed twice, and inoculated into DF salt medium supplemented with 3 mM ACC as a nitrogen source, followed by incubation at 30 ° C and 130 rpm for 5 days. As a control, a DF salt medium to which nothing was added was used as a nitrogen source, and the ACC deaminase activity was measured by measuring the absorbance at 600 nm to confirm the degree of increase in the cells grown in the medium to which ACC was added.
그 결과, DDP77 및 DDP398 균주에서 ACC만을 질소원으로 첨가한 DF 염 배지에서 생장할 수 있음을 확인하여, ACC 탈아미노효소 활성이 있음을 확인하였다(표 1).As a result, it was confirmed that the DDP77 and DDP398 strains could grow in DF salt medium supplemented with only ACC as a nitrogen source, confirming that they had ACC deaminase activity (Table 1).
실험예 2. 분리한 균주의 미네랄 가용화능Experimental Example 2. Mineral solubilization ability of the isolated strain
토양 내 미네랄을 가용화하면 식물 생장을 촉진시킬 수 있으므로, 분리 균주들의 미네랄 가용화능을 평가하기 위하여 배양 시간별 아연 가용화능, 탄산칼슘 가용화능, 규산마그네슘 가용화능을 측정하였다.Since mineral solubilization in soil can promote plant growth, in order to evaluate the mineral solubilization ability of the isolates, the zinc solubilization ability, calcium carbonate solubilization ability, and magnesium silicate solubilization ability were measured for each culture time.
구체적으로, 인산 가용화능은 0.5% 칼슘 포스페이트(calcium phosphate)가 첨가된 고체 PVK(pikopvskaya) 아가(agar) 배지(0.05% yeast extract, 1% glucose, 0.5% calcium phosphate, 0.05% ammonium sulphate, 0.02% potassium chloride, 0.01% magnesium sulphate, 0.00001% manganese sulphate, 0.00001% ferrous sulphate 1.5% agar) 중앙을 코르크 보러(cork borer)로 잘라낸 뒤, 순수 분리된 균주의 상등액을 10 ㎕씩 접종하였다. 접종 후 30℃에서 4일간 배양하면서 배지에 클리어 존(clear zone)의 형성 유무를 조사하여 난용성 인산의 가용화능을 판정하였다Specifically, the phosphoric acid solubilization ability was measured in a solid PVK (pikopvskaya) agar medium (0.05% yeast extract, 1% glucose, 0.5% calcium phosphate, 0.05% ammonium sulphate, 0.02% calcium phosphate, 0.02% calcium phosphate). potassium chloride, 0.01% magnesium sulphate, 0.00001% manganese sulphate, 0.00001% ferrous sulphate 1.5% agar) After cutting the center with a cork borer, 10 μl of the supernatant of the pure separated strain was inoculated. After inoculation, the solubilization ability of poorly soluble phosphoric acid was determined by examining the formation of a clear zone in the medium while culturing at 30 ° C for 4 days.
아연 가용화능은 Tris-minimal salt(1% D-glucose, 0.606% Tris-HCl, 0.468 % NaCl, 0.149% KCl, 0.107% NH4Cl, 0.043% Na2SO4, 0.02% MgCl2·2H2O, 0.003% CaCl2·2H2O, 1.5% agar, pH 7.0) 배지에 각 0.1244% ZnO, 0.1998% Zn3(PO4)2·4H2O, 0.1728% ZnCO3을 첨가하여 각 산화아연(ZnO), 인산아연(Zn3(PO4)2·4H2O), 탄산아연(ZnCO3)에 대한 가용화능을 확인하고자 하였다. 고체배지 중앙에 코르크 보러를 이용하여 잘라낸 뒤 순수 분리된 전배양 분리주를 접종 후 30℃에서 4일간 배양하면서 균체주위의 클리어 존(clear zone)의 형성 유무를 조사하여 ZnO, Zn3(PO4)2·4H2O, ZnCO3 가용화능을 측정하였다.Zinc solubilization was measured with Tris-minimal salt (1% D-glucose, 0.606% Tris-HCl, 0.468% NaCl, 0.149% KCl, 0.107% NH 4 Cl, 0.043% Na 2 SO 4 , 0.02% MgCl 2 2H 2 O , 0.003% CaCl 2 2H 2 O, 1.5% agar, pH 7.0) by adding 0.1244% ZnO, 0.1998% Zn 3 (PO 4 ) 2 4H 2 O, 0.1728% ZnCO 3 to each zinc oxide (ZnO ), zinc phosphate (Zn 3 (PO 4 ) 2 4H 2 O), zinc carbonate (ZnCO 3 ) solubilization ability wanted to check. After cutting with a cork borer in the center of the solid medium, inoculate the pure separated pre-cultured isolate, and then incubate at 30 ° C for 4 days, examining the formation of a clear zone around the cells to obtain ZnO, Zn 3 (PO 4 ) 2 ·4H 2 O , ZnCO 3 Solubilizing ability was measured.
탄산칼슘 가용화능은 Deveze-Bruni agar(0.5% D-glucose, 0.1% yeast extract, 0.1% peptone, 0.04% K2HPO4, 0.001% MgSO4, 0.5% NaCl, 0.005% (NH4)2SO4, 0.5% CaCO3, 1.5% agar, pH 6.8) 배지를 이용하였다. 고체배지 중앙에 코르크 보러를 이용하여 잘라낸 뒤 순수 분리된 전배양 분리주를 접종 후 30℃에서 4일간 배양하면서 균체주위의 clear zone 형성 유무를 조사하여 CaCO3 가용화능을 측정하였다.Calcium carbonate solubilization ability was measured in Deveze-Bruni agar (0.5% D-glucose, 0.1% yeast extract, 0.1% peptone, 0.04% K 2 HPO 4 , 0.001% MgSO 4 , 0.5% NaCl, 0.005% (NH 4 ) 2 SO 4 , 0.5% CaCO 3 , 1.5% agar, pH 6.8) medium was used. After cutting with a cork borer in the center of the solid medium, the pure separated pre-cultured isolate was inoculated and incubated at 30 ° C for 4 days, and the presence or absence of a clear zone around the cells was examined to measure the CaCO 3 solubilization ability.
규소 가용화능을 조사하기 위해서 silicate agar(1% D-glucose, 0.25% magnesium trisilicate, 1.5% agar, pH 6.8) 배지를 이용하였다. 고체배지 중앙에 코르크 보러를 이용하여 잘라낸 뒤 순수 분리된 전배양 분리주를 접종 후 30℃에서 4일간 배양하면서 균체주위의 clear zone 형성 유무를 조사하여 규소 가용화능을 측정하였다.To investigate the silicon solubilizing ability, a silicate agar (1% D-glucose, 0.25% magnesium trisilicate, 1.5% agar, pH 6.8) medium was used. The silicon solubilizing ability was measured by examining the formation of a clear zone around the cells while culturing at 30 ℃ for 4 days after inoculating the pure separated pre-cultured isolate after cutting it out using a cork borer in the center of the solid medium.
그 결과, DDP77 및 DDP398 균주는 공통적으로 인산 가용화능, 인산아연 가용화능, 탄산칼슘 가용화능 및 규산 마그네슘 가용화 활성을 가짐을 확인하였으며, DDP398 균주의 경우에는 인산 가용화능이 더 높은 활성을 나타내었다(도 2a 내지 도 2d 및 표 2).As a result, it was confirmed that strains DDP77 and DDP398 had phosphate solubilization ability, zinc phosphate solubilization ability, calcium carbonate solubilization ability and magnesium silicate solubilization activity in common, and in the case of strain DDP398, phosphate solubilization ability showed higher activity (Fig. 2a to 2d and Table 2).
++: 강한 미네랄 가용화능 활성; +: 중간 정도의 미네랄 가용화능 활성; -: 활성이 없음++: strong mineral solubilization activity; +: moderate mineral solubilizing activity; -: no activity
실험예 3. 분리한 균주의 세포외 효소 활성 확인Experimental Example 3. Confirmation of extracellular enzyme activity of the isolated strain
아밀라아제(amylase), 셀룰라아제(cellulase), 프로테아제(protease) 및 자일레나아제(xylanase)의 효소는 식물 병원성 곰팡이의 세포벽을 분해시키는 용균작용(degradative parasitism)을 수행해 균주가 식물병 방제 활성을 갖도록 하므로, 분리 균주들의 세포외 효소 활성도를 측정하였다.Enzymes such as amylase, cellulase, protease and xylanase perform degradative parasitism to degrade the cell wall of plant pathogenic fungi so that the strain has plant disease control activity. The extracellular enzyme activity of the isolated strains was measured.
구체적으로, 아밀라아제(amylase)활성의 경우 녹말이 함유된 영양 배지에 균주를 접종하여 30℃에서 2일간 배양한 후 원심분리(3000rpm, 10분)하였다. 배양 상등액과 1%의 녹말 용액(50mM 인산나트륨(sodium phosphate) 완충액)을 1:1로 혼합하여 20℃에서 3분간 반응시킨 후 발색 시약 용액(color reagent solution)을 혼합하여 100℃에서 15분간 열처리하였다. 아이스를 이용하여 냉각시킨 후 분광광도계(spectrophotometer, Multiskan GO, ThermoScientific, Vantaa, Finland)를 이용하여 540nm에서 흡광도를 측정하였다. Specifically, in the case of amylase activity, the strain was inoculated into a nutrient medium containing starch, incubated at 30 ° C. for 2 days, and then centrifuged (3000 rpm, 10 minutes). The culture supernatant and 1% starch solution (50 mM sodium phosphate buffer) were mixed 1:1 and reacted at 20 ° C for 3 minutes, and then the color reagent solution was mixed and heat-treated at 100 ° C for 15 minutes did After cooling with ice, absorbance was measured at 540 nm using a spectrophotometer (Multiskan GO, ThermoScientific, Vantaa, Finland).
효소 활성은 상기의 시험조건 하에서 녹말로부터 1 mg의 디-말토오스(D-maltose)에 상응하는 환원당을 생성하는 효소량으로 환산하여 나타냈다. The enzyme activity was expressed in terms of the amount of enzyme that produced reducing sugar corresponding to 1 mg of D-maltose from starch under the above test conditions.
셀룰라아제(cellulase)활성의 경우 카복시메틸셀룰로오스(carboxymethyl cellulose, CMC)를 함유한 영양 배지에 균주를 접종하여 30℃에서 2일간 배양한 후 원심분리(3000rpm, 10분)하였다. 원심분리한 상등액은 1%의 CMC(50mM 인산나트륨(sodium phosphate) 완충액)를 혼합하여 50℃에서 10분간 반응시킨 후, 3,5-디니트로살리실산(3,5-dinitrosalicylic acid, DNS)시약을 첨가하여 100℃에서 10분간 열처리 후 냉각시켰다. 흡광도는 분광광도계(spectrophotometer, Multiskan GO, Thermo Scientific, Vantaa, Finland)를 이용하여 540nm에서 측정하였다.In the case of cellulase activity, the strain was inoculated into a nutrient medium containing carboxymethyl cellulose (CMC), incubated at 30 ° C for 2 days, and then centrifuged (3000 rpm, 10 minutes). The centrifuged supernatant was mixed with 1% CMC (50 mM sodium phosphate buffer) and reacted at 50 ° C for 10 minutes, followed by 3,5-dinitrosalicylic acid (DNS) reagent. It was added and cooled after heat treatment at 100 ° C. for 10 minutes. Absorbance was measured at 540 nm using a spectrophotometer (Multiskan GO, Thermo Scientific, Vantaa, Finland).
효소 활성은 상기의 시험조건 하에서 CMC로부터 1 mg의 포도당(glucose)에 상응하는 환원당을 생성하는 효소량으로 환산하여 나타냈다. The enzyme activity was expressed in terms of the amount of enzyme that produced reducing sugar corresponding to 1 mg of glucose from CMC under the above test conditions.
프로테아제(protease)의 활성의 경우 탈지유(skim milk)가 함유된 영양 배지에 균주를 접종하여 30℃에서 2일간 배양한 후 원심분리(3000rpm, 10분)하였다. 원심분리한 상등액은 0.65%의 카제인(50mM 인산나트륨(sodium phosphate) 완충액)을 혼합하여 37℃에서 10분간 반응시켰다. 그 후 0.44M의 트리클로로아세트산 (trichloroacetic acid)을 첨가하여 37℃에서 30분간 단백질을 응고시켰다. 응고된 단백질을 0.45μm 필터를 이용하여 여과한 후, 여과액과 3배 희석된 폴린-시오칼토 페놀 시약(Folin-ciocalteu's phenol reagent)을 혼합하여 37℃에서 30분간 반응시켰다. 흡광도는 분광광도계(spectrophotometer, Multiskan GO, Thermo Scientific, Vantaa, Finland)를 이용하여 660nm에서 측정하였다. In the case of protease activity, the strain was inoculated into a nutrient medium containing skim milk, incubated at 30 ° C. for 2 days, and then centrifuged (3000 rpm, 10 minutes). The centrifuged supernatant was mixed with 0.65% casein (50 mM sodium phosphate buffer) and reacted at 37° C. for 10 minutes. After that, 0.44M of trichloroacetic acid was added and the protein was coagulated at 37°C for 30 minutes. After filtering the coagulated protein using a 0.45 μm filter, the filtrate and 3-fold diluted Folin-ciocalteu's phenol reagent were mixed and reacted at 37° C. for 30 minutes. Absorbance was measured at 660 nm using a spectrophotometer (Multiskan GO, Thermo Scientific, Vantaa, Finland).
효소 활성은 상기의 시험조건 하에서 카제인으로부터 1 mg의 티로신(tyrosine)을 유리시키는 효소량으로 환산하여 나타냈다. The enzyme activity was expressed in terms of the amount of enzyme that liberated 1 mg of tyrosine from casein under the above test conditions.
자일레나아제(xylanase)의 활성의 경우 너도밤나무 자일란(beechwood xylan)을 함유한 영양 배지에 균주를 접종하여 30℃에서 2일간 배양한 후 원심분리(3000rpm, 10분)하였다. 원심분리한 상등액은 1%의 자일란(50mM 아세트산(acetic acid) 완충액)을 혼합하여 50℃에서 10분간 반응시켰다. 반응시킨 후 3,5-디니트로살리실산(3,5-dinitrosalicylic acid, DNS)시약을 첨가하여 100℃에서 10분간 열처리 후 냉각시켰다. 효소 활성은 상기의 시험조건 하에서 자일란으로부터 1 mg의 디-자일로오스(D-xylose)에 상응하는 환원당을 생성하는 효소량으로 환산하여 나타냈다.In the case of xylanase activity, the strain was inoculated into a nutrient medium containing beechwood xylan, incubated at 30 ° C. for 2 days, and then centrifuged (3000 rpm, 10 minutes). The centrifuged supernatant was mixed with 1% xylan (50 mM acetic acid buffer) and reacted at 50° C. for 10 minutes. After the reaction, 3,5-dinitrosalicylic acid (DNS) reagent was added, heat-treated at 100° C. for 10 minutes, and then cooled. The enzyme activity was expressed in terms of the amount of enzyme that produced reducing sugar corresponding to 1 mg of D-xylose from xylan under the above test conditions.
흡광도는 분광광도계(spectrophotometer, Multiskan GO, Thermo Scientific, Vantaa, Finland)를 이용하여 540nm에서 측정하였다. Absorbance was measured at 540 nm using a spectrophotometer (Multiskan GO, Thermo Scientific, Vantaa, Finland).
그 결과, DDP77 균주는 각각 0.18 mg/mL, 0.08 mg/mL, 0.58 mg/mL, 0.13 mg /mL의 아밀라아제, 셀룰라아제, 프로테아제, 자일레나아제의 활성을 나타내었으며, DDP398 균주는 각각 0.18 mg/mL, 0.09 mg/mL, 0.85 mg/mL, 0.16 mg/mL의 아밀라아제, 셀룰라아제, 프로테아제, 자일레나아제의 활성을 나타내었다(표 3).As a result, the DDP77 strain showed activities of 0.18 mg/mL, 0.08 mg/mL, 0.58 mg/mL, and 0.13 mg/mL of amylase, cellulase, protease, and xylenase, respectively, and strain DDP398 showed activities of 0.18 mg/mL, respectively. , 0.09 mg/mL, 0.85 mg/mL, and 0.16 mg/mL of amylase, cellulase, protease, and xylenase activities were shown (Table 3).
상기 결과에 따라서, 식물생장촉진, 미네랄 가용화능 및 세포외 효소 활성까지 고려하여 DDP398 균주를 최종적으로 선별하였다. According to the above results, the DDP398 strain was finally selected in consideration of plant growth promotion, mineral solubilization ability, and extracellular enzyme activity.
실험예 4. DDP398 균주의 생장조건 확인Experimental Example 4. Confirmation of growth conditions of DDP398 strain
상기 실험예 1 내지 4를 통하여 항진균 활성, 질소 고정화능, IAA 생성능, 사이드로포어 생성능, ACC 탈아미노효소 활성 및 세포외 효소 활성을 갖는 DDP398 균주를 선별하여 다양한 온도, pH, 염 농도에서의 생육능을 확인하였다.Through Experimental Examples 1 to 4, DDP398 strains having antifungal activity, nitrogen fixation ability, IAA generating ability, siderophore generating ability, ACC deaminase activity and extracellular enzyme activity were selected for growth at various temperatures, pH, and salt concentrations. ability was confirmed.
그 결과, DDP398은 10-35℃의 온도에서 생육하는 것을 확인하였으며, pH의 경우에는 5-11 범위에서 균주가 성장할 수 있음을 확인하였다. 또한, 염 농도 범위(NaCl %)에서는 0-10% 범위에서 균주가 성장할 수 있음을 확인하였다(표 4).As a result, it was confirmed that DDP398 grew at a temperature of 10-35 ° C., and it was confirmed that the strain could grow in the pH range of 5-11. In addition, it was confirmed that the strain could grow in the range of 0-10% in the salt concentration range (NaCl %) (Table 4).
실험예 5. Experimental Example 5. Microbacterium testaceum Microbacterium testaceum DDP398 균주의 동정Identification of strain DDP398
상기 실험예 1에서 선별한 DDP398 균주의 분류학적 동정을 위하여 16S rRNA 염기서열 분석을 수행하였다.The DDP398 strain selected in Experimental Example 1 For taxonomic identification, 16S rRNA sequencing was performed.
5-1. DDP398 균주의 16S rRNA 유전자 구간의 분리 및 정제5-1. Isolation and purification of the 16S rRNA gene segment of strain DDP398
분리된 균주의 균체를 TSB 배지에 접종하여 30℃에서 24시간 동안 배양한 후 원심분리하여 균체를 회수하였다. DNA 추출 키트를 이용하여 회수한 균체로부터 게놈 DNA를 추출하고, 추출된 DNA를 주형으로하여 중합효소 연쇄반응(polymerase chain reaction, PCR)을 수행하였다. 2.5 유닛(unit)의 DNA 중합효소(i-StarTaqTM DNA polymerase, iNtRON Biotechnology, 대한민국), DNA 시료 5 ㎕, 2.5 mM의 dNTPs 각 4.0 ㎕, 10 pmol의 프라이머(primer), 버퍼(pyrobest buffer) 5.0 ㎕, 및 멸균 증류수를 혼합하여 총 부피가 50 ㎕가 되도록 하였다. 프라이머는 하기 표 5의 범용 프라이머(universal primer)를 사용하였고, 표 6의 반응 조건으로 PCR을 수행하였다.Cells of the separated strain were inoculated into TSB medium, cultured at 30° C. for 24 hours, and centrifuged to recover cells. Genomic DNA was extracted from the recovered cells using a DNA extraction kit, and a polymerase chain reaction (PCR) was performed using the extracted DNA as a template. 2.5 units of DNA polymerase (i-StarTaqTM DNA polymerase, iNtRON Biotechnology, Korea), 5 μl of DNA sample, 4.0 μl each of 2.5 mM dNTPs, 10 pmol of primer, 5.0 μl of buffer , and sterile distilled water to a total volume of 50 μl. As primers, universal primers in Table 5 were used, and PCR was performed under the reaction conditions in Table 6.
증폭된 PCR 산물을 EtBr(ethidium bromide)로 15분 동안 염색한 후, 1.0% 아가로스 젤(agarose gel)에서 확인하였으며, PCR 정제 키트(Millipore PCR cleanup kit, Millipore, 미국)를 이용하여 정제하였다.The amplified PCR product was stained with EtBr (ethidium bromide) for 15 minutes, checked on a 1.0% agarose gel, and purified using a PCR purification kit (Millipore PCR cleanup kit, Millipore, USA).
5-2. 16S rRNA 염기서열 분석을 통한 DDP398 균주의 동정5-2. Identification of DDP398 strain through 16S rRNA sequencing
정제된 유전자의 염기서열과 기존의 세균과의 유사성을 NCBI의 BLAST를 이용하여 분석하였고, MEGA 7.0 프로그램의 NJ(Neighbor-joining) 알고리즘을 이용하여 계통분석을 수행한 후 계통도(도 3)를 작성하였으며, 이 때 부트스트랩핑(bootstrapping)은 2,000 회 반복하였다.The similarity between the nucleotide sequence of the purified gene and the existing bacteria was analyzed using NCBI's BLAST, and a phylogeny was created using the MEGA 7.0 program's NJ (Neighbor-joining) algorithm, followed by phylogenetic analysis At this time, bootstrapping was repeated 2,000 times.
그 결과, DDP398 균주는 Microbacterium testaceum 균주와 99%의 상동성을 가지는 것으로 나타났다. 이에, DDP398 균주를 Microbacterium testaceum DDP398로 명명하고, 2021년 2월 23일자로 한국미생물보존센터에 수탁번호 KFCC 11880P로 기탁하였다.As a result, the DDP398 strain was found to have 99% homology with the Microbacterium testaceum strain. Accordingly, the DDP398 The strain was named Microbacterium testaceum DDP398, and on February 23, 2021, it was deposited with the Korea Microorganism Conservation Center under accession number KFCC 11880P.
5-3 마이크로박테리움 테스타세움(5-3 Microbacterium testaceum ( Microbacterium testaceumMicrobacterium testaceum ) DDP398 균주의 형태학적 특성 확인) Confirmation of morphological characteristics of strain DDP398
마이크로박테리움 테스타세움(Microbacterium testaceum) DDP398 균주를 주사전자 현미경(Scanning electron microscope, SEM)으로 촬영하여 형태학적 특성을 확인하였다.Microbacterium testaceum ( Microbacterium testaceum ) DDP398 strain was photographed with a scanning electron microscope (Scanning electron microscope, SEM) to confirm morphological characteristics.
그 결과, 마이크로박테리움 테스타세움(Microbacterium testaceum) DDP398 균주는 막대 모양인 간균(bacillus)의 형태를 가짐을 확인하였다(도 4).As a result, it was confirmed that the Microbacterium testaceum DDP398 strain had a rod-shaped bacillus form (FIG. 4).
실험예 6. 마이크로박테리움 테스타세움(Experimental Example 6. Microbacterium testaceum ( Microbacterium testaceumMicrobacterium testaceum )) DDP398 균주의 생리학적 특성 확인Confirmation of physiological characteristics of DDP398 strain
마이크로박테리움 테스타세움(Microbacterium testaceum) DDP398 균주의 생리학적 특성을 확인하기 위하여, 상기 균주의 19종 효소 활성을 키트(API ZYM kit, biomιrieux, 프랑스)를 이용하여 제조사의 설명서에 따라 측정하였다.In order to confirm the physiological characteristics of the Microbacterium testaceum DDP398 strain, 19 enzyme activities of the strain were measured using a kit (API ZYM kit, biomιrieux, France) according to the manufacturer's instructions.
그 결과, 마이크로박테리움 테스타세움(Microbacterium testaceum) DDP398 균주에서 Alkaline phosphatase, Esterase(C4), Esterase(C8), Leucine arylamidase, Valine arylamidase, Crystine arylamidase, α-chymotrypsin, Acid phosphatase, Naphtol-AS-BI-phosphohydrolase, α-glucosidase, β-glucuronidase, α-glucosidase 및 β-glucosidase 효소 활성을 나타났으며, 그 중에서도 Leucine arylamidase와 α-glucosidase에서 강한 효소 활성을 나타내었다(표 7).As a result, Microbacterium testaceum ( Microbacterium testaceum ) DDP398 Alkaline phosphatase, Esterase (C4), Esterase (C8), Leucine arylamidase, Valine arylamidase, Crystine arylamidase, α-chymotrypsin, Acid phosphatase, Naphtol-AS-BI-phosphohydrolase, α-glucosidase, β-glucuronidase, α-glucosidase in strains and β-glucosidase enzyme activities, among which strong enzyme activities were shown in leucine arylamidase and α-glucosidase (Table 7).
+++ : 강한 효소 활성, ++: 중간 정도의 효소 활성, +: 효소 활성; -: 효소 활성 없음+++: strong enzymatic activity, ++: moderate enzymatic activity, +: enzymatic activity; -: no enzyme activity
<110> ANGEL, CO.,LTD. <120> Microbacterium testaceum DDP398 having activities of promoting plant growth and controlling plant disease, and uses thereof <130> 2021P-02-029 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1391 <212> DNA <213> Artificial Sequence <220> <223> 16s rRNA of Microbacterium testaceum DDP398 <400> 1 gcagtcgaac ggtgaagcca agcttgcttg gtggatcagt ggcgaacggg tgagtaacac 60 gtgagcaacc tgccctggac tctgggataa gcgctggaaa cggcgtctaa tactggatat 120 gagcccctat cgcatggtgg gggttggaaa gattttttgg tctgggatgg gctcgcggcc 180 tatcagcttg ttggtgaggt aatggctcac caaggcgtcg acgggtagcc ggcctgagag 240 ggtgaccggc cacactggga ctgagacacg gcccagactc ctacgggagg cagcagtggg 300 gaatattgca caatgggcga aagcctgatg cagcaacgcc gcgtgaggga tgacggcctt 360 cgggttgtaa acctctttta gcagggaaga agcgaaagtg acggtacctg cagaaaaagc 420 gccggctaac tacgtgccag cagccgcggt aatacgtagg gcgcaagcgt tatccggaat 480 tattgggcgt aaagagctcg taggcggttt gtcgcgtctg ctgtgaaatc ccgaggctca 540 acctcgggcc tgcagtgggt acgggcagac tagagtgcgg taggggagat tggaattcct 600 ggtgtagcgg tggaatgcgc agatatcagg aggaacaccg atggcgaagg cagatctctg 660 ggccgtaact gacgctgagg agcgaaaggg tggggagcaa acaggcttag ataccctggt 720 agtccacccc gtaaacgttg ggaactagtt gtggggacca ttccacggtt tccgtgacgc 780 agctaacgca ttaagttccc cgcctgggga gtacggccgc aaggctaaaa ctcaaaggaa 840 ttgacgggga cccgcacaag cggcggagca tgcggattaa ttcgatgcaa cgcgaagaac 900 cttaccaagg cttgacatat acgagaacgg gccagaaatg gtcaactctt tggacactcg 960 taaacaggtg gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc 1020 aacgagcgca accctcgttc tatgttgcca gcacgtaatg gtgggaactc atgggatact 1080 gccggggtca actcggagga aggtggggat gacgtcaaat catcatgccc cttatgtctt 1140 gggcttcacg catgctacaa tggccggtac aaagggctgc aataccgtga ggtggagcga 1200 atcccaaaaa gccggtccca gttcggattg aggtctgcaa ctcgacctca tgaagtcgga 1260 gtcgctagta atcgcagatc agcaacgctg cggtgaatac gttcccgggt cttgtacaca 1320 ccgcccgtca agtcatgaaa gtcggtaaca cctgaagccg gtggcccaac ccttgtggag 1380 gagccgtcga a 1391 <110> ANGEL, CO., LTD. <120> Microbacterium testaceum DDP398 having activities of promoting plant growth and controlling plant disease, and uses its <130> 2021P-02-029 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1391 <212> DNA <213> artificial sequence <220> <223> 16s rRNA of Microbacterium testaceum DDP398 <400> 1 gcagtcgaac ggtgaagcca agcttgcttg gtggatcagt ggcgaacggg tgagtaacac 60 gtgagcaacc tgccctggac tctgggataa gcgctggaaa cggcgtctaa tactggatat 120 gagcccctat cgcatggtgg gggttggaaa gattttttgg tctgggatgg gctcgcggcc 180 tatcagcttg ttggtgaggt aatggctcac caaggcgtcg acgggtagcc ggcctgagag 240 ggtgaccggc cacactggga ctgagacacg gcccagactc ctacgggagg cagcagtggg 300 gaatattgca caatgggcga aagcctgatg cagcaacgcc gcgtgaggga tgacggcctt 360 cgggttgtaa acctctttta gcagggaaga agcgaaagtg acggtacctg cagaaaaagc 420 gccggctaac tacgtgccag cagccgcggt aatacgtagg gcgcaagcgt tatccggaat 480 tattgggcgt aaagagctcg taggcggttt gtcgcgtctg ctgtgaaatc ccgaggctca 540 acctcgggcc tgcagtgggt acgggcagac tagagtgcgg taggggagat tggaattcct 600 ggtgtagcgg tggaatgcgc agatatcagg aggaacaccg atggcgaagg cagatctctg 660 ggccgtaact gacgctgagg agcgaaaggg tggggagcaa acaggcttag ataccctggt 720 agtccacccc gtaaacgttg ggaactagtt gtggggacca ttccacggtt tccgtgacgc 780 agctaacgca ttaagttccc cgcctgggga gtacggccgc aaggctaaaa ctcaaaggaa 840 ttgacgggga cccgcacaag cggcggagca tgcggattaa ttcgatgcaa cgcgaagaac 900 cttaccaagg cttgacatat acgagaacgg gccagaaatg gtcaactctt tggacactcg 960 taaacaggtg gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc 1020 aacgagcgca accctcgttc tatgttgcca gcacgtaatg gtgggaactc atgggatact 1080 gccggggtca actcggagga aggtggggat gacgtcaaat catcatgccc cttatgtctt 1140 gggcttcacg catgctacaa tggccggtac aaagggctgc aataccgtga ggtggagcga 1200 atcccaaaaa gccggtccca gttcggattg aggtctgcaa ctcgacctca tgaagtcgga 1260 gtcgctagta atcgcagatc agcaacgctg cggtgaatac gttcccgggt cttgtacaca 1320 ccgcccgtca agtcatgaaa gtcggtaaca cctgaagccg gtggcccaac ccttgtggag 1380 gagccgtcga a 1391
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