농업생명과학연구 45(6) pp.151-162 Journal of Agriculture & Life Science 45(6) pp.151-162 점박이응애, Tetranychus urticae (Acarina: Tetranychidae) 에대한약용식물추출물의살비효과 이정수 1 함은혜 2 추호렬 3 이석준 4 이동운 4,5* 1 식물보호연구소, 2 오상킨섹트, 3 경상대학교응용생물학과 ( 농업생명과학연구원 ), 4 경북대학교생태과학과, 5 경북대학교생태환경관광학부생물응용전공 Acaricidal Efficacy of Herbal Extracts against Tetranychus urticae (Acarina: Tetranychidae) Jung-Su Lee 1 Eun-hae Ham 2 Ho-Yul Choo 3 Suk-Jun Lee 4 Dong-Woon Lee 4,5* 1 Plant Protection Institute, Gimpo 415-802, Korea 2 Osangkinsect, Cheonan 330-872, Korea 3 Dept. of Applied Biology, Gyeongsang National Univ.(Insti. of Agri. and Life Sci.), Jinju 660-701, Korea 4 Department of Ecological Science, Kyungpook National Univ., Sangju 742-711, Korea 5 Major of Applied Biology, School of Ecological Environment and Tourism, Kyungpook National Univ., Sangju 742-711, Korea 초록 Received: OCT. 28. 2011, Revised: DEC. 13. 2011, Accepted: DEC. 20. 2011 점박이응애 (Tetranychus urticae) 에대한살비활성을가지는물질을알아보기위하여약용식물 25 과 35 종의메탄올또는헥산추출물을이용하여잎침지법과살포법으로실내와 pot, 야외에서실험을수행하였다. 살비활성은약용식물종류별로차이를보였다. 강남콩 (Phaseolus vulgaris var. humilis) 잎을 1,000 ppm 농도의추출물에 1 분간침지한후점박이응애에대한치사율을조사한결과, 비자나무 (Torreya nucifera) 열매추출물과팥꽃나무 (Daphne genkwa) 와도꼬마리 (Xanthium strumarium), 나팔꽃 (Pharbitis nil) 씨앗추출물이각각 56.8% 와 47.8, 47.7, 47.7% 의살비활성을나타내었다. 잎침지시간을 30 초로단축시켜점박이응애에대한살비효과검정시는 1 분침지에비하여효과가감소하였고, 나팔꽃과도꼬마리의살비활성이가장높았다. 도꼬마리와나팔꽃씨앗헥산추출물의반수치사농도는각각 1,824 ppm 과 1,899 ppm 이었다. 두식물의 1,000 ppm 온수및냉수추출물은점박이응애에대한살비효과가 20% 이하로낮았다. Pot 의강낭콩에점박이응애를접종한후분무법으로추출물을 1,000 ppm 농도로살포한결과도꼬마리와나팔꽃씨앗헥산추출물은각각 76.3 과 71.3% 의높은살비활성을나타내었다. 그리고온실에서국화의점박이응애를대상으로나팔꽃씨앗헥산추출물을 2,000 ppm 과 1,000 ppm 으로처리시에는 50.8% 와 35.1% 의방제가를보였다. 검색어 - 식물추출물, 점박이응애, 나팔꽃, 도꼬마리, 살비효과 ABSTRACT Methanol and hexane extracts from 35 species in 27 families of herbal plants were evaluated for their acaricidal activities against two-spotted spider mite, Tetranychus urticae by leaf-dipping and spraying methods in laboratory, pot and field, respectively. Acaricidal activities were different depending on herbal plants. When T. urticae was released on leaves of Phaseolus vulgaris var. humilis that were dipped in the 1,000 ppm hexane extracts from fruits of Torreya nucifer and Daphan genkwa, seeds of Xanthium strumarium and Pharbitis nil at one minute they experienced 56.8, 47.8, 47.7, and 47.7% mortalities, respectively. Dipping time influenced acaricidal activity, that is, one minute dipping was more effective than 30 second dipping of Phaseolus vulgaris var. humilis leaves. However, extracts of X. strumarium and P. nil seeds had comparatively high activities. Concentration of hexane extract of X. strumarium and P. nil seeds revealing LC 50 were 1,824 and 1,899 ppm, respectively. Extracts of both plants from hot and cold water were not effective *Corresponding author: Dong-Woon Lee Tel: +82-54-530-1212 Fax: +82-54-530-1218 E-mail: whitegrub@knu.ac.kr
152 Journal of Agriculture & Life Science 45(6) representing <20% mortality at 1,000 ppm. However, hexane extracts of X. strumarium and P. nil seeds were effective against T. urticae on P. vulgaris var. humilis representing 76.3 and 71.3% mortalities in pot, respectively. Control effects of hexane extract of P. nil seed were 50.8 and 35.1% at 2,000 and 1,000 ppm against T. urticae on Chrysanthemum morifolium in greenhouse, respectively. Key words - Plant extract, Tetranychus urticae, Pharbitis nil, Xanthium strumarium, Acaricidal activity Ⅰ. 서론점박이응애 (Tetranychus urticae) 는사과나무나배 나무와같은과수는물론, 콩이나목화와같은밭작물, 가지, 오이, 토마토등의채소류, 장미와같은화훼류, 더덕과같은약용작물, 클로버나별꽃, 쑥, 망초, 환삼덩쿨과같은수십종의잡초등넓은기주범위를가진세계적인주요해충이다 (Hall and Ferree, 1975; Stacey et al., 1985; Croft et al., 1987; Wilson et al., 1991; McNab and Jerier 1993; Song et al., 1995; Ho, 2000; Takefugi et al., 2000). 점박이응애의발생초기에는밀도가낮아피해증상이잘나타나지않으나식물체의조직을흡즙하여잎의표면에백색의작은반점이나타난다. 그러다가밀도가점차증가하면잎뒷면에성충과약충이무리지어가해하기때문에잎이작아지고기형이되며, 누렇게변하면서점차말라죽는피해를준다. 성충은 0.3~0.4 mm의소형으로 25 에서알에서성충까지 10일정도소요되어연 10세대이상발생한다 (Mitchell, 1973). 그러나온도가높아짐에따라발육속도가빨라지기때문에시설재배지에서는발생세대수가증가될수있다 (Kim et al., 2008). 한편, 점박이응애를방제하기위해다양한농약들을광범위하게사용하고있는데약제의오용과남용으로약제저항성발달문제가심각한해충의하나로우리나라에서도다양한농약과작물에서저항성발달이보고되고있다 (Kim & Lee, 1989; Kim et al., 1994; Cho et al., 1995; Song et al., 1995; Lee et al., 2003b). 따라서이러한약제저항성을극복하기위하여다양한연구들이수행되어왔다 (Cho et al., 1993; Kim et al., 1994; Park et al., 1995; Ahn et al., 1996, 2004; Lee et al., 2004; Yu et al., 2005a). 그러나이러한방법들은점박이응애의약제저항성발달문제를근본적으로해결할수없고, 환경문제가대두됨으로인해점박이응애의방제전략은생물적방제와약제저항성 관리를두축으로하는연구들이수행되고있다. 즉, 천적과농약을조화시켜상호보완적으로사용하거나재배관리까지도고려한해충종합관리체계의개념에서연구가이루어지고있다 (Croft et al., 1987; Ahn et al., 1996; Kim et al., 1999). 그러나대부분의환금작물병해충의경우와마찬가지로점박이응애에대해서도약제사용을완전히배제한경제적이고, 효과적인방제수단이정립되고있지는않는실정이다. Lee(1990) 와 Kim(1994) 은사과와녹차재배지에서포식성천적만으로는식식성응애류의개체군밀도를경제적피해수준이하로유지하기어렵다고한바있다. 이와같이식식성응애인점박이응애의방제를위한약제의사용이불가피한상황에서근래에는세계각국에서천적에는독성이낮으면서광범위한살비효과를보이는한편환경에는큰영향을주지않는식물체추출물연구에많은관심이집중되고있다 (Isman, 2000; Kim et al., 2000; Chiasson et al., 2001; Momen et al., 2001; Kim, 2005; Shi et al., 2006). 식물체추출물을이용한병해충생리활성물질의연구는다양한분야에서시도되고있는데, 점박이응애의경우도식물추출물이나정유의살비또는기피효과가연구되었다 (Hiremath et al., 1995; Chiasson et al., 2001; Lee et al., 2003a, 2005; Choi et al., 2004; Yu et al., 2005b). 이러한연구결과에의해식물정유를기반으로하는해충방제들이등장하고있으며 (Isman, 2000) 우리나라의경우도친환경농업육성법에의하여식물체유래물질을바탕으로한친환경유기농자재들이급증하고있다. 점박이응애는세계각국에서주요경제작물에많은피해를주기때문에식물자원을이용한대체적인방제법들이많이연구되고시도되었으나각자원식물에대한전반적인활성검정은자원식물들의종류에비하여부족한실정이다. 특히생약자원이풍부한우리나라의경우살비활성가능성이있는식물에대한지속적인
Lee et al. : Acaricidal Efficacy of Herbal Extracts against Tetranychus urticae (Acarina: Tetranychidae) 153 연구가필요하다. 이에본연구는살충이나살선충효과가있는것으로알려진한약재를선발하여일반경제작물은물론약초재배지에서도문제시되고있는점박이응애의방제가능성을알아보기위하여수행하였다. Ⅱ. 재료및방법 2.1 점박이응애실험에이용한점박이응애는경북문경시에소재한동그라미곤충농장 ( 주 ) 에서분양받은감수성계통으로실험실에서온도 25±1, 상대습도 60 70%, 광주기 16L:8D 의조건하에서약제처리없이누대사육하면서이용하였다. 점박이응애는원예용상토를담은 16 20 cm pot 에강낭콩 (Phaseolus vulgaris var. humilis) 을 10 립씩파종한후제1 본엽이완전히전개되었을때접종하여사육하였는데, pot에서의이탈을방지하기위해사각용기 (35 40 4 cm ) 에물을담고는 pot를넣어두었다. 그리고점박이응애를접종한후 1주일간격으로강낭콩 pot를추가로넣어주면서계속증식시켜실험에이용하였다. 2.2 식물체의선발및추출살비활성후보약용식물의선발은신농본초경, 명의별록, 본초강목, 동의보감등의서적에서살충또는살선충효과가있는것으로알려진 29종을선발하여 28종은경동시장 ( 서울시동대문 ) 에서구입하였으며 neem나무의잎은필리핀산을이용하였다. 또한살충효과가있다고구전되어온약용식물들중된장풀 (Desmodium caudatum) 은잎과줄기를 10월에제주도에서, 미치광이풀 (Scopolia parviflora) 의뿌리는 4월에강원도인제에서, 오동나무 (Paulownia coreana) 잎은 10월에경기도가평에서, 머위 (Petasites japonicus) 와은행나무 (Ginkgo biloba) 잎은경북상주에서 10월에, 멀구슬나무 (Melia azadrach) 열매는 10월에경남거제에서, 다닥냉이 (Lepidium apetalum) 는 5월에경북상주지역에서채집하여이용하였다. 멀구슬나무와다닥냉이는추출부위를달리하여시료를조제하였다. 수집된 27과 35종 37시료의약용식물들은 (Table 1) 실온에서 1-2 주간건조한후분쇄기 (Hanil HMF-370) 를이용하 여최종 300 μm이하로분말화한후메탄올, 핵산, 온수, 냉수추출법으로추출하여시료로사용하였다. 각식물체추출물들중 31시료는 Lee et al.(2011) 이사용한시료를이용하였으며, 인동덩굴과잇꽃의꽃, 머위와오동나무, 은행나무잎및미치광이풀뿌리는메탄올로추출 (Kim et al., 2003) 하여실험에이용하였다. 각시료들중뿌리나줄기, 잎등의부분은메탄올로추출하였고, 사상자와비자, 오수유, 멀구슬나무열매와도꼬마리와나팔꽃의씨와같은지방성분이많은시료들은헥산추출한시료를이용하였다 (Kim et al., 2003). 2.3 실내실험약용식물추출물의점박이응애에대한살비효과를조사하기위한실내실험은 Hiremath et al. (1995) 의방법에따라엽침지법으로실험하였다. 1차실험은각각의시료를 4 의냉장고에서꺼내어 1시간동안정치한후 0.05 g씩을 100 ml비이커에넣고, 증류수와 Triton X-100 을첨가하여 (0.1 ml /l) 1,000 ppm의현탁액으로만들어생물검정시료로사용하였다. 뚜껑중앙에직경 4 cm, 200 Mesh의망이부착되어내 외부의공기유통이가능하며습도조절이간편한직경 10 cm의 petri dish에증류수 2 ml를묻힌여과지 ( 직경 9 cm, Toyo filter No. 2, Toyo Roshi, Japan) 2장을깔았다. 그리고증류수를충분히적신탈지면을 petri dish주위에둘러서수분을공급하는동시에응애의이탈을방지하게하였다. 그런다음 1,000 ppm으로희석한추출물에파종후 10일된강낭콩의엽절편 ( 직경 3.7 cm ) 을 1분간침지시킨뒤, 강낭콩엽절편의뒷면이위로향하도록하여 1시간동안음건하였다. 음건시킨엽절편은 petri dish의중앙부분에올려놓은후점박이응애성충 20마리를미세한붓으로접종시켰다. 무처리는엽절편을 Triton X-100 만첨가한 (0.1 ml /l) 증류수에침지시켜이용하였다. 접종후온도 25±1, 상대습도 60-70%, 광주기 16L:8D 의항온항습기에보관하였다. 처리 48시간후에치사유무를조사하였으며 4반복으로실시하였다. 2차실험은 1차실험후살비활성이우수한 13종의약용식물추출물을증류수와 Triton X-100을첨가하여 (0.1 ml /l) 1,000 ppm으로희석시킨후수행
154 Journal of Agriculture & Life Science 45(6) Table 1. List of plant species used for methanol and hexane extraction and acaricidal activity of 1,000 ppm herbal extracts against Tetranychus urticae Family name Scientific name ** Korean name Used part % corrected mortality (mean ± SD) Araceae Acorus gramineus Solander 석창포 Ro 15.3±19.0 d-i *** Arecacea Areca catechu L. 빈랑 Se 13.5±14.3 e-i Aspidiaceae Dryopteris crassirhizoma Nakai 관중 Rh 38.7±3.1 a-f Asteraceae Carpesium abrotanoides L. 담배풀 Fr 29.7±21.6 a-i Carthamus tinctorius L. 잇꽃 Fl 36.9±8.3 a-g Auckylandia lappa L. 목향 Ro 0.9±3.1 j Petasites japonicus Max. 머위 L 9.9±12.5 g-i Xanthium strumarium L. * 도꼬마리 Fr 47.7±16.5 ab Caprifoliaceae Lonicera japonica Thunberg 인동덩굴 Fl 4.5±8.3 ij Combretaceae Quisqualis indica L. 사군자 Se 44.1±11.4 a-c Convolvulaceae Pharbitis nil Choisy * 나팔꽃 Se 47.7±12.5 ab Cruciferae Lepidium apetalum Willdenow 다닥냉이 Ro 13.5±10.8 d-i Lepidium apetalum Willdenow 다닥냉이 St 11.7±8.3 e-i Fabaceae Albizzia julibrissin Duraz 자귀나무 Ba 29.7±19.5 a-i Gentianaceae Gentiana scabra var. buergeri Max. 용담 Ro 27.9±16.5 a-i Ginkgoaceae Ginkgo biloba L. 은행나무 L 27.9±16.5 a-i Lauraceae Machgilus thunbergii Sieb. et Zucc 후박나무 Ba 13.5±14.3 e-i Leguminosae Desmodium caudatum Dc. 된장풀 L & St 38.7±6.2 a-f Sophora flacescens Aiton 고삼 Ro 18.9±5.4 b-i Liliaceae Aloe ferox 알로에 L 17.1±13.6 c-i Meliaceae Azadirachta indica Neem tree Se 42.3±13.6 a-d Melia azedarach L. 멀구슬나무 Ba 11.7±15.6 f-i Melia azedarach L. * 멀구슬나무 Se 44.1±3.1 a-c Myrtaceae Eugenia caryophyllata 정향나무 Fb 4.5±3.1 ij Polygonaceae Polygonum aviculare L. 마디풀 St 0.9±3.1 d-i Polygonum tinctorium Lour 쪽 St 17.1±29.8 j Rheum coreanum Nakai 장군풀 Ro 6.3±3.1 h-f Rosaceae Prunus mume Sieb. et Zucc. 매실나무 Fr 38.7±34.8 a-f Rotaceae Evodia officinalis Dode * 오수유나무 Fr 40.5±23.6 a-e Rubiaceae Rubia akane Nakai 꼭두서니 P 31.5±17.4 a-i Scrophulariaceae Paulownia coreana Uyeki 오동나무 L 0.9±3.1 j Simaroubaceae Ailanthus altissima Swingle 가중나무 Ba 45.9±9.4 a-c Solanaceae Scopolia parviflora (Dunn) Nakai 미치광이풀 Ro 0.9±3.1 j Taxaceae Torreya nucifera Sieb. et Zucc. * 비자나무 Se 56.8±9.4 a Thymelaeaceae Daphne genkwa Sieb. et Zucc. 팥꽃나무 Fb 47.8±11.3 ab Umbelliferae Torilis japonica (Houtt.) Dc. * 사상자 Se 27.9±16.5 a-i Zingiberaceae Zingiber officinale Roscoe 생강 Rh 45.9±14.3 a-c * Hexane extract. ** Fb, flower bud; Ba, bark; Fl, flower; Fr, fruit; L, leaf; P, pericarp; Rh, rhizome; Ro, root; Se, seed; St, stem. *** Means followed by the same letter are not significantly different P 0.05 by Duncan's multiple range test. Leaf disk was soaked in each 1,000ppm extract for 1 minute and mortality was checked 48 hours after treatment. 하였는데, 엽절편을시료에 30 초만침지하여효과를 검정하였고, 실험방법은 1 차실험과동일하였다. 무 처리는엽절편을 Triton X-100 만첨가한 (0.1 ml / l) 증류수에침지시켜수행하였으며 4 반복으로수
Lee et al. : Acaricidal Efficacy of Herbal Extracts against Tetranychus urticae (Acarina: Tetranychidae) 155 행하였다. 3차실험은 2차실험후살비활성이우수한나팔꽃과도꼬마리추출물을대상으로농도별 (10,000, 2,500, 1,000, 760, 500, 200, 100 ppm) 로실험을수행하였다. 실험방법은 1차실험과동일하게수행하였으며 4반복으로수행하였다. 4차실험은메탄올이나핵산추출시료로활성을검정한결과, 활성이높았던나팔꽃과도꼬마리의시료를대상으로온 냉수추출법으로추출한후수행하였다. 100배액으로추출한각각의온 냉수추출물 5 ml를 100 ml비이커에넣고, 증류수를첨가하여각각 1,000 ppm과 4,000 ppm으로희석시켜실험에사용하였다. 실험방법은 1차실험과동일하게수행하였으며 4반복으로수행하였다. 2.4 Pot 실험약용식물추출물의점박이응애에대한살비효과를조사하기위한 pot 실험은 Song et al. (1995) 의방법에따라분무법으로실험하였다. 1차실험은실내실험에서활성이높았던나팔꽃, 도꼬마리, 비자나무의 hexane 추출시료와대조구는친환경자재로시판되고있는 Na-no{( 주 ) 비아이지 } 를 1,000 ppm으로희석시켜실험을수행하였다. 한약재추출물은 Triton X-100 을 0.1 ml /l 농도로첨가하여사용하였다. 강낭콩을직경 6 cm, 높이 6 cm의 pot에 2립씩파종한뒤한포기만을제 2본엽이완전히전개될때까지키워서강낭콩의잎에점박이응애 20마리를접종한후 30분간정착시킨다음 1,000 ppm으로희석한시료를 30 cm거리에서 hand sprayer (Komax Co.) 로강낭콩잎이충분히적실정도로 5초동안분무하였다. 처리한 pot는항온항습기 ( 온도 25±1, 상대습도 60-70%, 광주기 16L :8D) 에 6일간넣어둔뒤점박이응애의치사유무를현미경하에서조사하였다. 무처리는 Triton X-100 만첨가한 (0.1 ml /l) 증류수를살포하였고, 한개의 pot를한반복으로하여 3반복으로수행하였다. 2.5 야외실험약용식물추출물의점박이응애에대한살비효과를조사하기위한야외실험은 2009년 6월 17일경남진주의경상대학교부속농장비닐하우스에서수행하 였다. 2009년 2월 10일국화 ( 품종 : 옐로우푸마 ) 를재식거리 20 cm 20 cm로삽목한후 6월 17일난괴법 3반복으로실험구를설정하였다. 각구에서 20엽에있는점박이응애의사전밀도를조사하였는데, 실험구는 3 m2크기로설정하였다. 처리는나팔꽃메탄올추출물 1,000 ppm과 2,000 ppm 및대조약제비페나제이트액상수화제를 1,000배액으로희석하여 20l 등짐분무기로살포하였는데, 약액이흘러내릴정도로고루살포하였다. 5일후각구의 20엽에있는점박이응애의수를조사하였다. 무처리는물만살포하였다. 2.6 통계분석실험의결과는처리평균간차이를 Duncan's multiple range test나 Tukey's studentized range test(proc ANOVA, SAS Institute, 1999) 로분산분석하였으며, 치사율은 arcsine 변환후분석하였다. 농도별치사율결과는 probit 분석으로반수치사량 (LD 50 ) 을구하였다 (PROC PROBIT, SAS Institute, 1999). 모든분석의결과는평균 ± 표준편차로표기하였다. Ⅲ. 결과 3.1 약용식물추출물의수율실험에이용한 37종의약용식물중본연구에서추출한 6종시료의수율은머위의경우 6.42% 였으며인동덩굴은 7.11%, 잇꽃은 1.93%, 은행나무잎은 4.76%, 오동나무잎은 8.2%, 미치광이풀은 7.13% 였다. 3.2 살비효과실내검정약용식물추출물을 1,000 ppm으로희석한용액에강낭콩엽절편을침지하여점박이응애성충의치사율을조사한결과, 비자나무 (Torreya nucifera) 추출물처리가 56.8% 로가장높았고, 팥꽃나무 (Daphne genkwa), 도꼬마리 (Xanthium strumarium), 나팔꽃 (Pharbitis nil) 이각각 47.8, 47.7, 47.7% 의치사율을나타내었으며, 가중나무 (Ailanthus altissima), 생강 (Zingiber officinale), 멀구슬나무 (Melia azedarach), 사군자 (Quisqualis indica), neem(azadirachta indica), 오수유나무 (Evodia rutaecarpa), 관중
156 Journal of Agriculture & Life Science 45(6) (Dryopteris crassirhizoma), 된장풀 (Desmodium caudatum) 등이 38.7-45.9% 로비교적높은치사율을 나타내었다 (Table 1)(df=37, 114, F=4.71, p= 0.0001). 1차실험후활성이높았던 13종의약용식물추출 물을대상으로엽절편의침지시간을달리하여 2차 실험을수행한결과, 도꼬마리와나팔꽃씨앗처리 에서각각 42.3% 의치사율을나타내었고, 비자나무는 26.9% 의치사율을나타내었으며나머지처리에서는 1 차 실험에서보다 낮은 치사율을 보였다 (Table 2)(df=13, 42, F=27.85, p=0.0001). 1차와 2차실험에서점박이응애에대한살비활성이우수한나팔꽃과도꼬마리의시료의농도별점박이응애에대한효과를조사한결과는 Table 3과같았다. 식물체추출물의점박이응애에대한반수치사농도는도꼬마리가 1,824 ppm으로나팔꽃의 1,899 ppm 과거의비슷하였고, LC 90 은나팔꽃씨앗이 13,190 ppm으로도꼬마리씨앗에비하여두배정도낮게나타났다. Table 2. Acaricidal activities of 1,000 ppm methanol or hexane extracts derived from 13 herbal plants against Tetranychus urticae 48h after using the leaf dipping method Family name Scientific name % mortality (mean ± SD) Aspidiaceae Dryopteris crassirhizoma 10.3±3.0 c * Asteraceae Xanthium strumarium 47.4±7.7 a Combretaceae Quisqualis indica 5.1±3.0 c Convolvulaceae Pharbitis nil 47.4±7.7 a Leguminosae Desmodium caudatum 2.6±0.0 cd Leguminosae Sophora flavescens 7.7±4.2 c Meliaceae Azadirachta indica 9.0±7.7 c Meliaceae Melia azedarach 3.8±2.6 cd Rotaceae Evodia rutaecarpa 7.7±4.2 c Simaroubaceae Ailanthus altissima 2.6±0.0 cd Taxaceae Torreya nucifera 26.9±4.9 b Thymelaeaceae Daphne genkwa 9.0±7.7 c Zingiberaceae Zingiber officinale 7.7±4.2 c Control - 0.0 d Leaf disks were soaked in 1,000 ppm herbal extracts for 30 seconds. * Means followed by the same letter are not significantly different P 0.05 by Tukey's studentized range test. Table 3. Value of lethal concentration (ppm) of herbal extracts against Tetranychus urticae determined by leaf dipping method in petri dish Plant species LC 50 (95 % fiducial limit) LC 90 (95 % fiducial limit) Xanthium strumarium 1,899 (983.9-5,777) 29,530 (8,348-953,744) Pharbitis nil 1,824 (673.3-17,601) 13,190 (3,646-24,929,852) Herbal plants were extracted by hexane. N=80. 메탄올과핵산추출시료로활성을검정한결과를토대로활성이우수한나팔꽃과도꼬마리씨앗을온수와냉수추출법으로추출한후 1,000 ppm에서치사율을조사한결과는 Fig. 1과같았다 (df=4, 15, F=5.49, p=0.00063). 온수와냉수추출물에대한점박이응애의치사율은 20% 이하로메탄올추출물 에비해현저히낮았다. 도꼬마리추출물의경우온수추출과냉수추출의두방법에따른점박이응애의치사율은차이를보이지않았으나, 나팔꽃의경우는냉수추출물의살비활성이온수추출물에비하여현저히낮게나타났다.
Lee et al. : Acaricidal Efficacy of Herbal Extracts against Tetranychus urticae (Acarina: Tetranychidae) 157 3.3 살비효과 pot 실험실내실험에서활성이높았던한약재를 1,000 ppm 으로희석시킨후분무법으로 pot처리한결과는 Fig. 2와같았다. 대조약재로사용한 Na-no 는점박이응 애에대하여 100% 의치사율을보인반면도꼬마리, 나팔꽃은각각 76.3% 와 71.3% 의치사율을보여비교적높은결과를얻었고, 비자는 50.0% 의치사율을나타내었다 (df=4, 10, F=172.79, p=0.0001). Table 4. Efficacy of hexane extract of Pharbitis nil seed against Tetranychus urticae in Chrysanthemum morifolium on green house Treatment % survival (mean ± SD) Control efficacy (%) Pharbitis nil 2,000 ppm 59.7 ± 9.8 b * 50.8 Pharbitis nil 1,000 ppm 78.8 ± 3.4 c 35.1 Bifenazate 23.5 % SC 2.7 ± 4.8 a 97.8 Control 121.4 ± 14.9 d - * Means followed by the same letter are not significantly different P 0.05 by Tukey's studentized range test. Mortality was checked 5 days after treatment. 100 75 Hot Cold 50 25 0 b b ab P. nil X. strumarium Fig. 1. Effect of 1,000 ppm herbal extracts on Tetranychus urticae mortality depending on extraction methods (cold and hot water extracts) of Pharbitis nil and Xanthium strumarium in petri dish. Bars indicate standard deviation of the mean. The same letter over the bars in each treatment indicates that there is no significant difference among means (p<0.05). a Fig. 2. Effect of 1,000 ppm herbal extracts, Pharbitis nil, Torreya nucifera and Xanthium strumarium and agricultural friendly materials on Tetranychus urticae using spraying method in pots. Bars indicate standard deviation of the mean. The same letter over the bars in each treatment indicates that there is no significant difference among means (p<0.05).
158 Journal of Agriculture & Life Science 45(6) 3.4 살비효과야외실험국화에서점박이응애에대한나팔꽃씨앗헥산추출물의효과를야외에서조사한결과, 2,000 ppm처리에서는 50.8% 의방제가를보였으며 1,000 ppm농도에서는 35.1% 의방제가를보였다 (df=3, 8, F=131.42, p=0.0001)(table 4). Ⅳ. 고찰 실험에이용한시료들중팥꽃나무, 도꼬마리, 나팔꽃씨앗은점박이응애에대한활성이처음으로확인되었다. 이들중팥꽃나무의경우동일속에속하는서향 (Daphan odora) 으로부터살선충활성물질인 odoracin 과 odoratrin 이알려진바있으나 (Prakash and Rao, 1997) 응애류에대한활성물질에대해서는알려진바없다. 도꼬마리는감기나두통, 류머티스, 관절염, 해열및발한등의치료제로우리나라를비롯하여아시아여러국가에서사용하고있는데 (Huang, 1993; Bae, 2000) 항암이나항염증활성등이알려져있고, 일부 xanthanolide 와같은물질들이분리되었다 (Saxena & Mondal, 1994; Mahmoud, 1998; Ma et al., 1998; Kim et al., 2005b). 또한도꼬마리추출물은식물병원성진균에대하여항진균활성이있는것으로알려져있는데 (Kim et al., 2002; Park et al., 2005), Kim et al. (2002) 은 4-oxo-1 (5), 2,11, (13)-xanthatriene-12,8-olide 이항균활성을가지는물질이라고하였다. 나팔꽃씨앗은 flavonoid, anthocyanin, deterpenoid, triterpen saponin, phenolic amide 등의다양한물질들이분리되었는데항암이나항균작용등의약리활성에대한연구들만주로수행되었고 (Jung et al., 2008; Kim et al., 2010, 2011), 점박이응애를비롯한해충에대한활성을가지는물질단계의연구들은일부식물체에서만연구되고있는실정이다. 된장풀은제주지역에서구더기방제용으로민간에서이용되어지는식물인데점박이응애에대해서도활성이비교적높게나타났다. 생약재추출물을이용한점박이응애활성검정은몇몇의연구진들에의해수행된바있는데본연구에서이용한소재와중복되는부분도있었지만효과는상이하게나타났다. Kim et al.(2005a) 은 1,000 ppm의농도로희석한 후박나무잎과, 도둑놈의지팡이뿌리, 인동덩굴의잎과줄기의추출물을 petri dish에서분무법으로검정하여각각 58.8, 80.1, 86.7% 의치사율을얻었고, Park et al.(2002) 은후박나무잎추출물을 2,000 ppm의농도로침지법으로검정한바있는데점박이응애성충에대한치사율이 50% 이상이라고하였다. 그러나본실험에서는후박나무수피추출물은 13.5% 의치사율을보였으며도둑놈의지팡이뿌리는 18.9%, 인동덩굴꽃은 4.5% 의치사율을보였다. 후박나무나인동덩굴의경우추출부위가달라효과에서차이가있었던것으로보인다. 실제본실험의멀구슬나무에서도수피와열매는활성에서차이가있었다. Hiremath et al.(1995) 도추출부위에따라점박이응애에대한활성이다르게나타난다고하였다. 이러한차이는식물체의부위에따라구성물질의성분에서차이가나기때문이다 (Singh et al., 2010). 한편, 도둑놈의지팡이는동일부분의추출물을이용하였지만점박이응애에대한영향에서 4배이상의차이가났다. 이는사용한시료의원산지차이에의한것이거나처리방법에의한차이로생각된다. 생강추출물도점박이응애에효과가있는것으로알려져있는데 Lee et al.(2001) 은생강추출물을 2,000 ppm으로희석시켜비닐하우스내에서점박이응애에처리한결과 90% 이상의방제효과를얻었는데, 본실험에서는 1,000 ppm 농도에서 45.9% 의치사율을얻었다. 식물체추출물의경우식물체의채취시기나가공상태에따라약효가다르게나타날수있다. 따라서생강의경우추후부위별활성검정이나채취시기, 수집장소에따른활성검정이추가로수행되어져야할것으로생각된다. 은행잎은약리작용뿐만아니라일부해충에대한살충작용도있는것으로알려져있는식물체로 bilobalide와 ginkgolide A, B등이점박이응애에대해살비효과를가지는물질로알려져있다 (Lee et al., 2005). 그러나본실험에서는 8.1% 의낮은치사율을보였다. 이는추출방법의차이와살포방법및은행잎의채취시기등에의한차이로생각된다. Lee et al.(2005) 은열수추출한은행잎을사용하였다. 그결과푸른은행잎추출물은 200 ppm의분무법검정에서 80.7% 의살비효과를보였고, 100 ppm에서는 98.3% 의살비율을보였으며노란은행잎추출물의살
Lee et al. : Acaricidal Efficacy of Herbal Extracts against Tetranychus urticae (Acarina: Tetranychidae) 159 비율은매우낮게나타났었다. 석창포는이집트숲모기나토고숲모기, 빨간집모기의유충과같은위생곤충에대한살충활성이있는물질로 (Yang et al., 2004) 알려져있지만점박이응애에서는낮은치사율을보였다. 용담 ( 뿌리 ) 추출물 2,000 ppm은배추좀나방과담배거세미나방유충에대하여 80% 이상의살충율을나타내어나방류유충에대해활성이높은물질로보고되어있다 (Kwon et al., 1994). 본실험에서도점박이응애에대해비교적높은치사율을보였다. 그리고인동덩굴 ( 잎, 꽃 ) 의추출물은살충율은낮지만흰줄숲모기에대해기피효과가우수한것으로알려져있는데 (Kyong & Yoon, 2000) 점박이응애에서는낮은치사율을보였다. 기피활성유무에대해서는검정이필요할것으로생각된다. 도꼬마리와나팔꽃씨앗추출물을제외하고는, 대부분 1차에비하여 2차실험에서살비활성이낮게나타났다. 1차실험에서는엽절편의침지시간을 1분으로하였는데, 이경우일부처리에서엽의일부가수침이되는조직파괴현상이발견되어 2차실험에서는침지시간을 30초로단축시켰다. 따라서수침시간의차이가살비활성의차이를유발시킨것으로생각된다. 반면도꼬마리와나팔꽃씨앗추출물은침지시간의단축에도불구하고살비활성에서변화를보이지않아이후의실험에서는이두식물체를주로이용하여실험을수행하였다. 침지시간은활성성분의흡수와이행에영향을미치므로야외에서약재를살포할경우에도약액의살포량은성분의흡수와잔류에영향을미치는주요요인이다. 따라서야외적용성이높은것을선발하기위해서는가급적짧은시간의처리에서활성효과가높은것을선발하는것이바람직하다. 냉수와온수추출법은단시간내에다량의시료를확보할수있고, 특별한장치가필요없어농가에서쉽게활용할수있는추출방법이다. 따라서본연구에서는농가에서쉽게이용할수있고상업화시킬때비용을절감할수있는방법인냉수와온수추출법을이용하여점박이응애에대한활성을검정하였으나헥산추출물에비하여현저히낮은효과를보였다. 추출방법이나추출시사용하는용매의종류에따라서도서로다른성분이분리되기때문에생물활성이 다르게나타날수있다. Chiasson et al.(2001) 은 Artemisia absinthium 이나 Tanacetum vulgare 등은추출방법에따라점박이응애에대한활성에차이가있을뿐만아니라함유하고있는성분의종류나양에도차이가있다고하였다. Pot 실험에서나팔꽃씨추출물농도에따른점박이응애에대한살비활성은실내실험에비하여높은치사율을보였다. 이는처리방법의차이에의한것으로생각된다. 즉, 실내실험에서는엽침지법을사용하였으나 pot와야외실험에서는분무법을이용하였기때문에약제의충체에대한직접접촉이많아짐으로인하여치사율이높아진것으로생각된다. 그럼에도불구하고야외실험에서방제가는 50.8% 로난황유 (Park et al., 2008) 나포식성천적인칠레이리응애 (Phytoseiulus persimilis) 의방사때 (Moon et al., 2006) 보다낮게나타나실용적인측면에서는고려해볼결과였다. 그러나경제성이있는범위내에서높은효과를보이는농도를탐색하거나살비효과를보이는유효성분을규명하여그들을이용한방제연구를수행한다면좀더실용적인이용이가능할것으로생각된다. Ⅴ. 감사의글 실내와야외실험수행에많은도움을준경상대학교선충실험실실원들과경북대학교잔디해충및선충실험실실원들에감사를표한다. 식물체추출에기술적도움을준권순열연구원에게도감사를표한다. 이논문은농림수산식품부농림기술개발사업의지원에의해수행된결과이다. Literature Cited Ahn, Y. J., J. K. Yoo, J. R. Cho, J. O. Moon, and S. C. Lee. 1996. Evaluation of effectiveness of AC303630 and flucycloxuron mixture against Tetranychus urticae (Acari: Tetranychidae) under laboratory and field conditions. Appl. Entomol. Zool. 31: 67-73. Ahn, K. S., S. Y. Lee, K. Y. Lee, Y. S. Lee, and G. H. Kim. 2004. Selective toxicity of pesticides to the
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