미생물,발효,생물공학 : Burkhoderia sp. AK-17에 의한 잎들깨 재배의 생물학적 조절

김근기 ( Keun Ki Kim ),김용균 ( Yong Kyun Kim ),손홍주 ( Hong Joo Son ),최영환 ( Young Whan Choi ),강규영 ( Kyu Young Kang ) 한국응용생명화학회 2005 Applied Biological Chemistry (Appl Biol Chem) Vol.48 No.1

Characterization of an antifungal compound isolated from an antagonistic fungus Aspergillus terreus against phytopathogenic fungi

Keun-Ki Kim(김근기),Jae-Gon Kang(강재곤),Yong-Lark Choi(최용락),Han-Dae Yun(윤한대),Ho-Sung Ha(하호성),Kyu-Young Kang(강규영) 한국농약과학회 1998 농약과학회지 Vol.2 No.1

Aspergillus terreus로부터 항진균성 물질의 분리 및 구조분석

김근기,박기훈,문석식,강규영 ( Keun Ki kim,Ki Hun Park,Suk sik Moon,Kyu Young Kang ) 한국응용생명화학회 1997 Applied Biological Chemistry (Appl Biol Chem) Vol.40 No.6

In the course of search antagonistic fungi from soil in green house, four kind of fungi (AF1, AF2, AF3, AF4) were isolated, which have activities aganist Phytophthora capsici, Botrytis cinera, Rhizoctomia solani, Pythium ultimum and Fusarium oxysporum. The AF2 was identified according to the morphological description of Aspergillus terreus. This antagonistic fungus inhibiting various plant pathogens was effective to reduce disease incidence of cucumber seedlings caused by mixed inoculum of Rhizoctonia solani, Pythium ultimum and Fusarium oxysporum. Antifungal compound I was isolated and purified by fresh chromatography from A. terreus. The ¹H and ^(13)C assignment of compound I was achieved from two-dimensional ¹H-¹H COSY, HMQC, HMBC with the add of homonuclear and heteronuclear double resonance experiment. The compound I was identified butyrolactone I (α-oxo-β-(p-hydroxyphenyl)-γ-(p-hydroxy-m-3,3-dimethyl-allylbenzyl)-γ-methoxy-carbonyl-γ-butyrolactone, C_(24)H_(24)O_7, M.W.=424).

Aspergillus terreus로부터 항진균성 물질의 분리 및 구조분석

김근기,박기훈,문석식,강규영,Kim, Keun-Ki,Park, Ki-Hun,Moon, Suk-Sik,Kang, Kyu-Young 한국응용생명화학회 1997 Applied Biological Chemistry (Appl Biol Chem) Vol.44 No.4

In the course of search antagonistic fungi from soil in green house, four kind of fungi (AF1, AF2, AF3, AF4) were isolated, which have activities against Phytophthora capsici, Botrytis cinera, Rhizoctonia solani, Pythium ultimum and Fusarium oxysporum. The AF2 was identified according to the morphological description of Aspergillus terreus. This antagonistic fungus inhibiting various plant pathogens was effective to reduce disease incidence of cucumber seedlings caused by mixed inoculum of Rhizoctonia solani, Pythium ultimum and Fusarium oxysporum. Antifungal compound I was isolated and purified by fresh chromatography from A. terreus. The $^1H$ and $^{13}C$ assignment of compound I was achieved from two-dimensional $^1H-^1H\;COSY$, HMQC, HMBC with the add of homonuclear and heteronuclear double resonance experiment. The compound I was identified butyrolactone I (${\alpha}$-oxo-${\beta}$-(p-hydroxyphenyl)-${\gamma}$-(p-hydroxy-m-3,3-dimethyl-allylbenzyl)-${\gamma}$-methoxycarbonyl-${\gamma}$-butyrolactone, $C_{24}H_{24}O_7$, M.W.=424). 시설내 토양으로부터 식물병원균에 활성을 갖는 길항균을 탐색하는 과정에서 주요 식물 병원균인 Phytophthora capsici, Botrytis cinerea, Rhizoctonia solani, Pythium ultimum 및 Fusarium oxysporum에 활성을 갖는 4종류의 길항균(AF1, AE2, AE3, AF4)을 분리하였다. 이들 중 가장 높은 활성을 나타낸 AE2균을 동정한 결과, 생육한 균은 짙은 오렌지색과 황갈색을 띄었으며, 현미경의 morphology는 column형의 conidial head와 foot cell로 부터 곧게 뻗은 분생자와 2중층의 포자낭병을 형성했다. 그리고 MY20 agar 배지에서 투명한 둥근세포의 형성등이 Aspergillus terreus의 형태와 일치 하였다. Pot에서 병원균과 A. terreus를 동시에 처리하여 오이의 생육을 조사한 결과, 토양중량당 A. terreus의 균체를 1% 처리에 40%, 5% 처리에 57%, 10% 처리에는 75% 병발생율을 억제시켰다. 그리고 A. terreus의 배양여액으로부터 activity-guided fractionation을 실시하여 항진균성 물질인 화합물 I 을 얻었다. $^1H$, $^{13}C\;NMR$, DEPT, $^1H-^1H\;COSY$, HMQC, HMBC 및 질량스텍트럼을 분석한 결과 화합물 I 은 butyrolactone I (${\alpha}$-oxo-${\beta}$-(p-hydroxyphenyl)-${\gamma}$-(p-hydroxy-m-3,3-dimethyl-allylbenzyl)-${\gamma}$-methoxycarbonyl-${\gamma}$-butyrolactone, $C_{24}H_{24}O_7$, M.W. = 424)로 동정되었다.

Effect of Phytohormones and Chemical Inhibitors on Pathogenesis-related Genes Identified by Differential Hybridization in Rice Suspension Culture Cells

김상곤,Jingni Wu,Ethan E White,Young Whan Choi,Keun-Ki Kim,In Soo Choi,Sun-Hyung Kim,강규영,김선태,Yiming Wang,김용철 한국식물병리학회 2010 Plant Pathology Journal Vol.26 No.4

In order to study disease resistance mechanisms in rice against the rice blast fungus Magnaporthe grisea, we screened fungal elicitor-responsive genes from rice suspension-cultured cells treated with fungal elicitors employing differential hybridization (DH). By DH screening,31 distinct rice clones were isolated and a majority of them were full-length cDNAs encoding pathogenesisrelated (PR) genes. Sixteen of the 31 genes were upregulated at 4, 8, and 12 h following fungal elicitor treatment. To elucidate the effect of signal molecules and biotic elicitors on the regulation of rice defense genes, we further characterized the transcriptional expression patterns of representative isolated PR genes;OsGlu1, OsGlu2, OsTLP, OsRLK, and OsPR-10, following treatment with fungal elicitor, phytohormones, cycloheximide,and inhibitors of protein phosphorylation. Jasmonic acid (JA) induced transcriptional expression of OsGlu1, OsTLP, and OsRLK, but not of OsGlu2 and OsPR-10 at any of the tested time points. Salicylic acid (SA) and abscisic acid weakly induced the expression of OsTLP and OsRLK. SA showed an antagonistic effect with fungal elicitor and JA. Cycloheximide suppressed all these genes upon elicitor treatment, except for OsGlu2. Staurosporine only induced the expression of OsRLK. Application of calyculin A strongly induced OsRLK expression, but suppressed the expression of OsGlu2. Our study yielded a number of PR genes that play a role in defense mechanisms against the rice blast fungus, as well as contribute towards the elucidation of crosstalk between phytohormones and other modifications during defense signaling

Burkhoderia sp. AK-17에 의한 잎들깨 재배의 생물학적 조절

김근기,김용균,손홍주,최영환,강규영 Plant molecular biology and biotechnology research 2005 Plant molecular biology and biotechnology research Vol.2005 No.

There are various crop diseases in green houses that are caused by the cultural environments, especially high temperature and moisture. To solve the forementioned problems, farmers are overusing agricultural chemicals, causing other damages by the chemical residue. In this study, antagonistic bacteria as biological control agents were isolated to produce the environmentally-friendly crops for use in green houses. Eighteen species of antagonistic bacteria were totally isolated from the soil and plants in the Perilla fields, and AK-17 showed the highest activity among the isolates. According to the results of anti-fungal spectrum against several pathogens by AK-17, the antagonism effect of the isolates was remarkable against grey mold rot by Botrytis cinerea, sclerotinia rot by Sclerotnia sclerotiorum, and stem rot by Rhizoctonia solini. To evaluate the biological control effects of the isolates against the major diseases of Perilla, studies were carried out to evaluate the preventive and the curative effects of the diseases throughout the pot experiments. According to the forementioned experiments, the preventive and the curative effects by the isolates against sclerotinia rot were respectively showed as 55% and 92%. For the grey mold rot, those were 40% and 78%, respectively. As to the evaluation of the growth-promoting effect by AK-17, the length and the biomass of the tested plants were increased to 120% and to 164%, respectively. For the leaf numbers and area were respectively increased to 120% and 220%. Furthermore, AK-17 was identified as Burkhoderia sp. according to the results of physiological properties and genetic methods. 시설원예단지의 재배환경은 고온다습으로 많은 병해가 발생하며, 이를 방제하기위해 과다한 농약을 사용하므로 농약 잔류성에 대한 피해가 심각한 우려를 낳고 있다. 미생물을 이용한 생물학적 방제법 개발로 청정 시설원예작물을 생산할 목적으로 길항균을 분리하였다. 잎들깨 재배토양과 식물체로부터 길항세균 18종을 분리하였으며 분리한 길항세균 중에 AK-17이 가장 활성이 뛰어나 이를 이용하여 주요 식물병원균에 대한 항균스펙트럼을 조사하였다. 그 결과 잿빛곰팡이병원균의 Botrytis cinerea와 균핵병원균의 Sclerotinia sclerotiorum 및 줄기썩음병원균인 Rhizoctonia solant에 대한 항균효과가 뛰어났다. 잎들깨의 주요 병에 대한 생물학적 방제실험은 병발생 억제효과와 병방제효과를 pot 실험으로 실시했다. 그 결과 균핵병은 55%의 병발생 억제효과와 92%의 방제효과가 있었고, 잿빛곰팡이병은 40%의 병발생 억제효과와 78%의 방제효과를 확인할 수 있었다. AK-17의 식물 생육촉진효과는 신장이 120%, 생체중이 164% 증가되었으며, 엽수와 엽면적은 각각 120%와 220%의 증가효과를 보였다. 그리고 AK-17을 생리·생화학적방법과 유전학적 방법으로 동정한 결과, Burkhoderia sp.로 확인되었다.

Gliocladium virens G872로부터 항균물질의 분리 및 특성

강규영,김한수,김근기,손홍주,이영근,김용균 密陽産業大學校 農業技術開發硏究所 1999 農業技術開發硏究所報 Vol.3 No.1

토양 근권의 길항성 미생물인 Gliocladium virens G872가 분비하는 항균물질은 R. solani, F. oxysporum에 대해 활성이 있는 viridin과 P. ultimum에 활성이 있는 gliovirin 등을 TLC, silicagel column chromtography 및 TLC plate direct bioassay방법에 의해 분리, 확인하였다. 그 외에도 새로운 unknown compound를 배양여액(Un 1)과 균체(Un 2, Un 3)로 부터 3종을 분리 하였으며, 균배양에 사용한 배지에 따라 항균물질의 생성 유무와 생성량의 차이가 있었다. PDB나 CDM에서는 virgin이 생성되며, CDM에서는 viridin 외에 또 다른 항균물질(Un 1)이 생성되었다. 그 반면 SS liquid medium에서는 항균물질이 생성되지 않았다. 배양여액으로부터 분리한 새로운 항균물질인 Un 1, M은 주요식물병원군인 P ultimum, R. solani, F. oxysporum의 전 균에 강력한 항균활성을 뛰었고, 균체에서 분리한 항균물질 Un 3, M은 P. ultimum과 R. solani에 항균력이 뛰어났다. UR 1과 Un 3의 항균작용은 병원균의 균사를 lysis시키는 항균 작용을 보였다. Un 1, M의 기기분석 결과, 분자량 390의 dioctyl phthalate (??)로 확실시된다. Gliocladium virens G872 was establish, survive and protiferate in rhizosphere, and induce plant growth. The plant growth promotion is accomplished by the control of minor pathogens. The impact of control of minor pathogens is currently being explored. G. virens G872 had been elucidated as antibiotisis by overproduction of antibiotics such as viridin, gliovirin, and gliotoxin together with yet unidentified 3 more antibiotics from culture suspension and cell mat. The antibiotics were isolated and identification by thin-layer choromatography, in situ bioassay, petriplate bioassay, and instrumental analysis. The UV absorption of Un 1, M showed at 214nm and shoulder appeared at 289 nm. IH-NMR spectra was showed at 0.7-2 ppm which indicate hydrocarbon chain and 2.1-2.5 ppm. GC/MS spectra was showed molecular ion peak at m/e 390. These instrumental analysis allow us cautiously predict the presence of diotyl phthalate. G. virens G872 was produced the antifungal proteins from the minimal culture containing cell mat of pathogens. Growth responses induced by G. virens G872 appear to be due to the control of pathogens or deleterious pathogens.

초피(Zanthoxylum piperitum) 용매추출물의 항균활성

손병구,강규영,김용균,손홍주,김한수,김근기,최영환 密陽産業大學校 農業技術開發硏究所 1998 農業技術開發硏究所報 Vol.2 No.1

초피나무(Z. piperitum)를 6개 부분(과피, 종자, 잎, 목피, 목질부, 뿌리)으로 나누어서 순차적용매 추출법(Hexane, Chloroform, Ethylacetate, Methanol, H₂O)을 이용하여 각 각의 추출물을 얻었다. 각 추출물은 주요 식중독 원인균인 S. aureus, S. typhimurium, V. parahemolyticus, E. coli 0157:H7에 대해 항균력을 조사하였다. 과피의 용매추출물에서는 gram positive bacteria와 gram negative bacteria 모두에 항균력을 나타냈고, 그 중에서도 Hexane, Chloroform, 그리고 Ethylacetate 추출물은 S. aureus, V. parahemolyticus, E. coli O157:H7에 항균력을 보였으며, Methanol 추출물은 V. parahemolyticus에 아주 강력한 항균활성을 나타냈다. 종자, 잎, 그리고 목피의 용매 추출물은 S. aureus, V. parahemolyticus에만 항균 활성을 뛰었고, 종자의 Hexane 추출물은 S. aureus 에 아주 강력한 항균 활성을 뛰었다. 목질부의 Hexane, Chloroform, Ethylacetate 추출물은 gram positive bacteria에만 항균활성을 뛰었고, Methanol 추출물은 E. coli 0157: H7, V. parahemolyticus. S. typhimurium 즉 gram negative bacteria에만 항균성을 뛰는 특이성을 보였다. 뿌리에서도 methanol 추출물만이 gram negative bacteria에 항균력을 나타냈다. 부위와 추출용매에 따라 항균작용 스펙트럼에 특이성을 보였다. Escherchia coli 0157:H7 Salmonella typhimurium, Vibrio parahemolyticus. and Styphylococcus aureus were pathogens involved in food poisoning in numerous countries. The inhibitory effects of plants, collected from miryang district, on the growth of food Poison bacterials, were investigated. Among Zanthoxylum piperitum was remarkably effective to inhibit the growth of the strains. An antibiotic substances were extracted by hexane, chloroform, ethylacetate, methanol, and water from pericarp, seed, leaf, bark, wood and root of Z. piperitum. Generally, the organic solvent extracts were exhibited stronger antibacterial activity against all the bacteria tested. The ethylacetate extract and methanol extract of the pericarp were strongest growth inhibition activity against S. aureus and V parahemolytieus, respectively, and the hexane extract of the seed was too The methanol extract of the wood had strongest growth inhibition activity against gram-negative bacteria such as E. coli O157:H7, V. parahemolyticus, and S. typhimurium, and other oraganic extracts showed the gram-positive bacteria.