Isolation, Screening, and Molecular Identification of Streptomyces sp. W-200 and Its Bioherbicidal Activity in Weed Control

조광민,신승철,Aung Bo Bo,UMURZOKOV MIRJALOL,WeiQiang Jia,박기웅,최정섭,김영숙 한국잔디학회 2022 Weed & Turfgrass Science Vol.11 No.3

A total of 332 bacterial strains were isolated and screened for bioherbicidal activity. The three isolates namely W12, W-177, and W-200 were found to be efficient in suppressing Digitaria ciliaris by 88.3, 89.67, and 95.5% as compared with untreated control. Among them, the isolate W-200 caused severe injury to the growth of D. ciliaris, eventually leading to plant death. Phylogenetic analysis of 16S rRNA sequencing determined that the isolate W-200 was 99% similar to Streptomyces formicae (1H-GS9). Streptomyces strain W-200 was tested on D. ciliaris through soil and foliage applications. There was a progressive reduction in the growth of D. ciliaris in both applications. Optimization of bacterial culture conditions showed that Streptomyces strain W-200 was intense bioherbicidal activity and high cell mass at 25℃, 150 rpm, and initial pH 7. The herbicidal activity against D. ciliaris in light conditions was slightly higher than that in dark conditions. The chlorophyll content decreased by 96, 65, and 61% after treatment with paraquat, Streptomyces strain W-200, and glufosinate-ammonium, respectively at x1 recommended concentration. Electrolyte leakage increased gradually with time until 24 hours after treatment of Streptomyces strain W-200. These results suggested that Streptomyces strain W-200 producing herbicidal activity may be a new bioherbicide candidate. A total of 332 bacterial strains were isolated and screened for bioherbicidal activity. The three isolates namely W12, W-177, and W-200 were found to be efficient in suppressing Digitaria ciliaris by 88.3, 89.67, and 95.5% as compared with untreated control. Among them, the isolate W-200 caused severe injury to the growth of D. ciliaris, eventually leading to plant death. Phylogenetic analysis of 16S rRNA sequencing determined that the isolate W-200 was 99% similar to Streptomyces formicae (1H-GS9). Streptomyces strain W-200 was tested on D. ciliaris through soil and foliage applications. There was a progressive reduction in the growth of D. ciliaris in both applications. Optimization of bacterial culture conditions showed that Streptomyces strain W-200 was intense bioherbicidal activity and high cell mass at 25℃, 150 rpm, and initial pH 7. The herbicidal activity against D. ciliaris in light conditions was slightly higher than that in dark conditions. The chlorophyll content decreased by 96, 65, and 61% after treatment with paraquat, Streptomyces strain W-200, and glufosinate-ammonium, respectively at x1 recommended concentration. Electrolyte leakage increased gradually with time until 24 hours after treatment of Streptomyces strain W-200. These results suggested that Streptomyces strain W-200 producing herbicidal activity may be a new bioherbicide candidate.

아미리스 정유의 제초활성

윤미선,김성문,조해미,최정섭,연보람 한국잔디학회 2012 Weed & Turfgrass Science Vol.1 No.4

본 연구의 목적은 아미리스 정유의 제초활성을 검정하는데 있다. 아미리스 정유의 기내 종자발아 제초활성 검정 결과 유채에 대한 GR50값은 8.8 μg g-1 이었으며, 온실제초활성 검정 결과 바랭이에 대한 완전고사 경엽처리 약량은 1,250 μg ml-1 이었다. 화본과잡초 4종과 광엽잡초 5종을 대상으로한 온실실험에서 4,000 μg ml-1 약량처리시아미리스 정유는 수수, 돌피, 자귀풀에 대해 각각 90%,70% 그리고 70%의 약효를 나타내었으며, 아미리스 정유에 처리된 잎은 모두 건조되는 증상을 나타내었다. 포장시험에서 아미리스 정유 5%의 피, 냉이, 토끼풀에 대한제초효과는 모두 70%이었으며, 10%에서는 100%이었다. 아미리스 10% 정유의 제초활성은 처리후 6시간부터 발현되기 시작하였으며, 아미리스 정유 처리구 잡초의 지상부위 생체중은 대조구와 비교하여 41.5%이었으나 지하부위생체중은 대조구와 비교하여 차이가 없었다. 아미리스 정유에는 총 15종의 화학물질이 검출되었으며 주된 화합물은 calarene(27.24%), elemol(19.38%), γ-eudesmol(9.24%),curcumene(5.85%), β-sesquiphellandrene(5.63%), zingiberene (5.50%), selina-3,7(11)-diene(5.29), 1,3-diisopropenyl-6-methylcyclohexene(4.18), β-bisabolene(4.13%), β-maaliene(3.62%)이 었다. 본 연구의 결과 아미리스 정유는 속효성, 비선택적, 비이행성 제초특성을 갖는 것으로 추론된다. The objective of this study was to know the herbicidal activity of the essential oil from amyris (Amyris balsamifera). In a seed bioassay experiment, the amyris essential oil inhibited the growth of rapeseed (Brassica napus)by fifty percent at 8.8 µg g-1. And in a greenhouse experiment, sorghum, barnyard grass and Indian jointvetch, which was applied in above-ground parts, with the amyris essential oil at 4,000 µg ml-1 showed visual injuries of 90, 70, and 70, respectively (0, no damage; 100, total damage). However, soil application of the essential oil did not show such herbicidal injuries. In a field experiment, foliar application of the amyris essential oil at 5% controlled effectively weeds such as barnyardgrass, shepherd's purse, and clover in 24 hours. Our results indicated that the amyris essential oil had herbicidal activity. To understand the composition of the amyris essential oil, the oil was analyzed by gas chromatography-mass spectometry with solid-phase micro-extraction apparatus. There were 15 organic chemicals in the oil and the major constituents were calarene, elemol, γ-eudesmol, curcumene, β-sesquiphellandrene, zingiberene, selina-3,7(11)-diene, 1,3-diisopropenyl-6-methyl-cyclohexene, β-bisabolene, and β-maaliene. Overall results suggest that the amyris essential oil had a herbicidal activity with fast, contact, and non-selective mechanism.

살초활성물질 함유 국내 자생식물의 탐색(V)

홍민지(Minji Hong),조남규(Nam-Kyu Cho),보셀라(Sela Bo),장혜정(Hyejeong Jang),황기환(Ki Hwan Hwang),손하라라잔 칸다사미(Kandhasamy Sowndhararajan),홍선희(Sun hee Hong),이용호(Yong Ho Lee),김성문(Songmun Kim) 한국잡초학회 2021 Weed & Turfgrass Science Vol.10 No.3

전세계의 제초제 개발회사에서는 새로운 작용기작을 갖는 신규 화합물을 식물로부터 탐색하고 있다. 본 연구의 목적은 살초활성이 있는 국내의 자생식물을 선발하는데 있다. 국내의 자생식물 63종 시료로부터 MeOH 조추출물을 얻은 다음 24-well tissue culture plate에서 유채(Brassica napus L.)와 나팔꽃(Ipomoea nil [L.] Roth)의 신초길이와 생체중을 측정하여 in vitro살초검정을 수행하였고, 온실에서 토양처리 및 경엽처리 후 다양한 광엽식물과 화본과식물의 잎에서 나타나는 살초증상을 달관평가하였다. 자생식물 63종 중 전동싸리와 흰전동싸리 등 29종은 유채에 대하여 중정도의 살초활성을 나타내었으며(1,000 μg<SUP>-1</SUP><GR50값<2,000 μg<SUP>-1</SUP>), 4종은 나팔꽃에 대하여 중정도의 살초활성을 나타내었다. 온실시험에서 추출물을 토양처리한 경우에는 살초효과가 전혀 발현되지 않았으나, 경엽처리의 경우, 금낭화, 말냉이, 소리쟁이, 참새귀리 추출물을 4 kg ai ha<SUP>-1</SUP> 약량으로 처리하였을 때 피의 잎이 뒤틀리면서 기형적으로 생장하였으며 생육이 억제되었다. 본 연구는 국내의 자생식물 중 살초활성을 나타낸 시료로부터 새로운 작용점을 갖는 살초활성물질을 탐색할 수 있을 것이다. Herbicide production companies around the world are searching for new compounds from plants with new modes of action. The objective of this study was to discover Korean native plants with herbicidal activity. After obtaining crude methanol extracts from 63 species of native plants in Korea, in vitro seed germination bioassay was performed by measuring shoot length and weight of rapeseed (Brassica napus L.) and morning glory (Ipomoea nil [L.] Roth) in 24-well plates. After the soil and foliage treatments under greenhouse conditions, the herbicidal symptoms were documented in the leaves of various broadleaf weeds and crops based on visual symptoms. Out of 63 species of Korean native plants, herbicidal activity was observed for 29 species against rapeseed (GR50 value of 1,000 to 2,000 μg<SUP>-1</SUP>) and 4 species against morning glory. In particular, Melilotus suaveolens and Melilotus albus exhibited moderate herbicidal activity against both rapeseed and morning glory. In the greenhouse experiment, the application of extracts on the soil did not show any herbicidal effect. In the case of foliage treatment (4 kg ai ha<SUP>-1</SUP>) of Dicentra spectabilis, Thlaspi arvense, Rumex crispus and Bromus japonicas, the leaves of Echinochloa crus-galli were twisted and deformed, and the growth was inhibited. The results revealed that plant samples are a potential source for the development of bioherbicides with new mechanisms of action.

제초성 Benzenesulfonyl urea계 화합물의 형태와 반응성

유성재,이상호,고영관,성낙도,Yu, Seong-Jae,Lee, Sang-Ho,Ko, Young-Kwan,Sung, Nak-Do 한국응용생명화학회 1996 Applied Biological Chemistry (Appl Biol Chem) Vol.44 No.1

The most stable stereo conformer in non substituted benzenesulfonyl urea, 1 was the II-keto form, which the molecule was intramolecular associated(H-bond) coformer between imide group and N atom on the Pyrimidine ring. The hydrolytic degradation of 2 derivatives were proceeds by nucleophilic addition reaction(p<0) with orbital controlled intermolecular interaction between LUMO with electron donating$(\sigma<0)$ groups of 2 and HOMO of water molecule. N-(4,6-disub. pyrimid ine-2-yl)aminocarbonyl-2-(1,1-dimethoxy-2-fluoro)ethylbenze nesulfonamides,3 and N-(4,6-disub. triazine-2-yl)aminocarbonyl-2-(1,1-d imethoxy-2-fluoro)ethylbenzenesulfonamides,4 we re synthesized and their herbicidal activities in vivo against bulrush (Scirpus juncoides.) were measured by the pot test under the paddy conditions And the structure activity relationships(SAR) were analyzed by the multiple regression technique. The results of the SAR suggested that the 3 and 4 derivatives indicated dependent on the hydrophobicity of the 4,6-disubstituents and the heterocyclo group, where the optimal value $((log\;P)_{opt.}=0.89)$ of hydrophobicity was 0.89. The pyrimidine substituents, 3 showed higher herbicidal activity than the triazine substituents, 4. Among them, 4,6-dimethoxypyrimidine substituent, 3a showed the best herbicidal activity. 제초성 비치환(H) benzenesulfonyl urea 분자, 1은 sulfonyl group 인접의 amino group과 pyrimidinyl group의 N원자 사이에 회합(H-결합)된 형태(II-keto)가 제일 안정하였으며 phenyl 치환$(R_2$ 및 $R_3)$유도체, 2는 전자를 밀수록$(\sigma<0)$ LUMO(ev.)는 음의 값으로 증가하여 물 분자의 HOMO(ev.)사이에 궤도 조절반응(p<0)에따른 전형적인 친핵 반응성을 나타낸다. N-(4,6-이치환-pyrimidine-2-yl)aminocarbonyl-2-(1,1-dimethoxy-2-fluoro)ethylbenzenesulfonamides, 3 및 N-(4,6-이치환-triazine-2-yl)aminocarbonyl-2-(1,1-dimethoxy-2-리uoro)ethylbenzenesulfonamide, 4 유도체의 4,6-이치환기(X 및 Y) 와 헤테로 고리의 변화에 따른 올챙이고랭이(Scirpus juncodies.) 에 대한 제초활성은 소수성$((log\;P)_{opt.}=0.89)$이 가장 큰 영향을 미치는 요인이었다. 또한, pyrimidine-치환체, 3이 triazine-치환체, 4보다 양호한 제초활성을 나타내었으며 dimethoxypyrimidine-치환체, 3a가 가장 큰 제초활성을 보였다.

1980년대 이후 우리나라 등록 제초제의 제반 특성별 현황

유진상(JinSang Yu),임태헌(TaeHeon Lim),최성환(SungHwan Choi),이동운(DongWoon Lee) 한국잡초학회·한국잔디학회 2020 Weed & Turfgrass Science Vol.9 No.1

우리나라에서 제초제의 공급은 1955년부터 시작하여 1970년대이후로 증가하고 있다. 본 조사는 1980년대 이후 우리나라에 등록된 제초제들의 일반적 현황(등록 품목, 독성별 구분, 제형별 구분, 성분별 구분)을 알아보기 위하여 수행하였다. 1982년이후 제초제 등록 품목수는 2014년을 정점으로 감소하는 경향이었다. 등록된 제초제의 85.3%가 저독성이었으며 2010년대 이후 등록된 제초제의 86.1%는 어독성 3급이었고, 1.9%만 어독성 1급이었다. 등록 된 제초제들의 제형별 구성은 1990년대 9종에서 2010년대 17종으로 다양성이 증가하였다. 1980년대 이후 등록된 제초제들의 18가지 제형들중 우점순위는 입제, 유제, 액제, 액상수화제 순이었는데 이들 네 제형의 점유비율은 76.2%였다. 등록 제초제들의 계통별 구성은 년대기별로 다양화되었으나 등록 품목수가 가장 많은 계통은 1990년대 이후 sulfonylurea계통이 가장 많았으며 두 번째는 chloroacetamide계통이었다. 전체 등록 제초제의 계통들 중 상위 3개 계통의 점유 비율은 년대기별로 감소하는 경향이었으나 평균 점유비율은 39.3%로 높았다. In Korea, the supply of herbicides began in 1955 and has increased since the 1970s. This study was conducted to find out the general status of herbicides registered in Korea since the 1980s (registration items, toxicity, formulation, and chemical component). Since 1982, the number of herbicide-registered items has declined since peaking in 2014. 85.3% of the registered herbicides were low toxicity, 86.1% of the herbicides registered since the 2010s were fish toxicity Ⅲ, and only 1.9% were fish toxicity Ⅰ. The formulation composition of registered herbicides increased from 9 in the 1990s to 17 in the 2010s. Among the 18 formulations of herbicides registered since the 1980s, the top priority was granules, emulsifible concentrate, soluble concentrate and suspension concentrate. The composition of registered herbicides varied according to the chronological age, but the sulfonylurea was the largest and the second was the chloroacetamide since the 1990s. The share of the top three lines of all herbicide lines declined by age, but the average share was high at 39.3%.

Response of Digitaria ciliaris and Cyperus spp. Populations to s-metolachlor and alachlor Herbicides in Korea

Umurzokov Mirjalol,Bo Aung Bo,박인곤,조광민,박상언,이증주 한국잔디학회 2024 Weed & Turfgrass Science Vol.13 No.1

This study examined how Digitaria ciliaris and Cyperus spp. populations in Korea respond to and develop herbicide resistance to s-metolachlor and alachlor. Seeds of D. ciliaris and Cyperus spp. were collected from 69 and 51 sites respectively, and a whole-plant doseresponse experiment was conducted in an experimental greenhouse. Nonlinear curve fit analysis elucidated GR50 values (the dose resulting in a 50% reduction in fresh weight) of D. ciliaris ranging from 38.3–124.6 g a.i. ha-1 for s-metolachlor and 23.6–38.2 g a.i. ha-1 for alachlor, resulting in a baseline sensitivity index (BSI) of 3.25 for s-metolachlor and 1.61 for alachlor. In the dose-response test with Cyperus spp., GR50 values ranged from 41.9–98.4 g a.i. ha-1 for s-metolachlor and 25.2–33.7 g a.i. ha-1 for alachlor, resulting in BSI values of 2.34 for s-metolachlor and 1.33 for alachlor. This study found no evidence of s-metolachlor and alachlor herbicide resistance in D. ciliaris and Cyperus spp. populations in Korea. However, regular monitoring and baseline sensitivity studies should be conducted to detect the potential evolution of herbicide-resistant weed populations.

A Phi Class Glutathione S-transferase from Oryza sativa (OsGSTF5): Molecular Cloning, Expression and Biochemical Characteristics

Cho, Hyun-Young,Lee, Hae-Joo,Kong, Kwang-Hoon Korean Society for Biochemistry and Molecular Biol 2007 Journal of biochemistry and molecular biology Vol.40 No.4

A glutathione S-transferase (GST) related to the phi (F) class of enzymes only found in plants has been cloned from the Oryza sativa. The GST cDNA was cloned by PCR using oligonucleotide primers based on the OsGSTF5 (GenBank Accession No. $\underline{AF309382}$) sequences. The cDNA was composed of a 669-bp open reading frame encoding for 223 amino acids. The deduced peptide of this gene shared on overall identity of 75% with other known phi class GST sequences. On the other hands, the OsGSTF5 sequence showed only 34% identity with the sequence of the OsGSTF3 cloned by our previous study (Cho et al., 2005). This gene was expressed in Escherichia coli with the pET vector system and the gene product was purified to homogeneity by GSH-Sepharose affinity column chromatography. The expressed OsGSTF5 formed a homo-dimer composed of 28 kDa subunit and its pI value was approximately 7.8. The expressed OsGSTF5 displayed glutathione conjugation activity toward 1-chloro-2,4-dinitrobenzene and 1,2-epoxy-3-(p-nitrophenoxy)propane and glutathione peroxidase activity toward cumene hydroperoxide. The OsGSTF5 also had high activities towards the herbicides alachlor, atrazine and metolachlor. The OsGSTF5 was highly sensitive to inhibition by S-hexylGSH, benastatin A and hematin. We propose from these results that the expressed OsGSTF5 is a phi class GST and appears to play a role in the conjugation of herbicide and GPOX activity.

Cloning of a rice tau class GST isozyme and characterization of its substrate specificity

Cho, Hyun-Young,Yoo, Sun-Young,Kong, Kwang-Hoon Elsevier 2006 Pesticide biochemistry and physiology Vol.86 No.2

<P><B>Abstract</B></P><P>The GST cDNA was successfully cloned from an <I>Oryza sativa</I> cDNA library by PCR using oligonucleotide primers based on the <I>Os</I>GSTU5 (GenBank Accession No. AF309377) sequence. The cDNA was composed of a 687-bp open reading frame encoding for 228 amino acids. The deduced amino acid sequence of this gene shared over 60% sequence identity with the sequences of the tau class <I>Zm</I>GSTU6 and <I>Zm</I>GSTU19. This gene was expressed in <I>Escherichia coli</I> with the pET vector system and the gene product was purified to homogeneity by GSH–Sepharose affinity column chromatography. The expressed <I>Os</I>GSTU5 formed a homo-dimer composed of 25kDa subunit and its p<I>I</I> value was approximately 7.5. The <I>Os</I>GSTU5 displays high activities towards 1-chloro-2,4-dinitrobenzene and 1,2-epoxy-3-(<I>p</I>-nitrophenoxy)propane. The activity of the <I>Os</I>GSTU5 was significantly inhibited by hematin and ethacrynic acid. The <I>Os</I>GSTU5 shows the highest activity towards chloro-<I>s</I>-triazine and acetanilide herbicides.</P>

A plant-specific tau class glutathione S-transferase from Oryza sativa with very high activity against 1-chloro-2,4-dinitrobenzene and chloroacetanilide herbicides

Jo, H.J.,Lee, J.J.,Kong, K.H. Academic Press 2011 Pesticide biochemistry and physiology Vol.101 No.3

A plant-specific tau class GST gene homolog was successfully cloned from an Oryza sativa cDNA library by PCR using oligonucleotide primers based on the OsGSTU4 (GenBank Accession No. AF309378) sequence. The cDNA was composed of a 720-bp open reading frame encoding 239 amino acids. The deduced amino acid sequence of this gene shared over 65% sequence identity with the sequences of the tau class TaGST28e45 and ZmGST42. Conversely, the OsGSTU4 sequence showed very low identity to the GST sequences of phi, theta and zeta classes. This gene was expressed in Escherichia coli with the pET vector system, and the gene product was purified to homogeneity using GSH-Sepharose affinity column chromatography. The expressed OsGSTU4 formed a homodimer with subunits of approximately 25.5kDa. OsGSTU4 displayed very high activity toward 1-chloro-2,4-dinitrobenzene. The activity of the OsGSTU4 was significantly inhibited by S-hexylglutathione and hematin. Plant OsGSTU4 had a unique herbicide specificity and played an important role in the detoxification reaction against fluorodifen and chloroacetanilide herbicides.

1-(4-chloro-2-fluoro-5-propargyloxyphenyl)-3-thiourea 유도체의 제초활성과 분자 유사성

성낙도,성민규,박관용,송종환 한국응용생명화학회 2008 Journal of Applied Biological Chemistry (J. Appl. Vol.51 No.3

In the search for third generation herbicidal cyclic imide derivatives, the average values of herbicidal activity (pI50) in vivo (pre-emergence) of 40 new peroxidizing herbicides, 1-(4-chloro-2-fluoro- 5-propargyloxyphenyl)thiourea derivatives (1-40) against rice plant (Orysa sativa) and barnyard grass (Echinochlor crus-galli) were studied. The molecular similarity between protoporphyrinogen IX (protogen) as the substrate of protox enzyme and Urea derivatives (1-40) was discussed quantitatively. The diallyl (20) and 3-nitro substituent (33) showed the selective herbicidal activity against barnyard grass. Allyl substituent (8) and their molecular similarity indice (S=0.81) showed the highest levels of herbicidal activity (pI50=4.71). Also, similarity indices (S) and superimposed volume (C) of protogen and aryl-substituents (21-40) showed good correlation. In the search for third generation herbicidal cyclic imide derivatives, the average values of herbicidal activity (pI50) in vivo (pre-emergence) of 40 new peroxidizing herbicides, 1-(4-chloro-2-fluoro- 5-propargyloxyphenyl)thiourea derivatives (1-40) against rice plant (Orysa sativa) and barnyard grass (Echinochlor crus-galli) were studied. The molecular similarity between protoporphyrinogen IX (protogen) as the substrate of protox enzyme and Urea derivatives (1-40) was discussed quantitatively. The diallyl (20) and 3-nitro substituent (33) showed the selective herbicidal activity against barnyard grass. Allyl substituent (8) and their molecular similarity indice (S=0.81) showed the highest levels of herbicidal activity (pI50=4.71). Also, similarity indices (S) and superimposed volume (C) of protogen and aryl-substituents (21-40) showed good correlation.