28 Journal of Agriculture & Life Science 46(2) Ⅰ. 서론시금치 (Spinacia olerecea L.) 는명아주과에속하 는 1년생저온성식물로고대페르시아가원산지로아랍상인들에의해인디아와중국으로전해져현재까지식용으로이용되는작물이다 (Vic

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1 농업생명과학연구 46(2) pp Journal of Agriculture & Life Science 46(2) pp 식물정유, 길항세균및난황유를이용한시금치노균병방제 이정한 1 정성우 2 전성식 3 강신권 3 최준민 3 임채신 4 고학룡 5 이광수 6 정남준 7 배동원 8* 1 경상대학교생명과학연구원, 2 경상대학교농업생명과학연구원, 3 한국국제대학교식품과학과, 4 경상남도농업기술원농업기술교육센터, 5 ( 재 ) 바이오 21 센터, 6 아이엠팜 ( 주 ), 7 ( 주 ) 남보, 8 경상대학교공동실험실습관 Control of Spinach Downy Mildew Using Essential Oil, Antagonistic Bacteria, Cooking Oil and Egg Yolk Mixture Jung-Han Lee 1 Sung-Woo Jeong 2 Sung-Sik Chun 3 Shin-Kwon Kang 3 Jun-Min Choi 3 Chae-Shin Lim 4 Hack-Ryong Ko 5 Kwang-Soo Lee 6 Nam-Jun Chung 7 Dong-Won Bae 8* 초록 1 Research Institute of Life Science, Gyeongsang National University, Jinju , Korea 2 Institute of Agriculture and Life Science, Gyeongsang National University, Jinju , Korea 3 Department of Food Science, International University of Korea, Jinju , Korea 4 Gyeongsangnam-do Agricultural Research and Extension Services, Jinju , Korea 5 Bio 21 Center, Jinju , Korea 6 IMFarm Co., Ltd., Jinju , Korea 7 Nambo Co., Ltd., Jinju , Korea 8 Central Instrument Facility, Gyeongsang National University, Jinju , Korea Received: DEC , Revised: APR , Accepted: APR 본연구는친환경적으로시금치노균병을방제하기위하여식물정유, 길항미생물및난황유의병발생억제효과를검정하여화학농약대신에이용할수있는친환경제제로서의가능성을검토하였다. 5 종의식물정유가운데노균병방제효과는 1 회처리구와 1 주간격 2 회처리구모두 Eucalyptus approximans 오일의병원균억제효과가 60% 정도로가장높은것으로나타났다. 반면에토양에서선발된길항균세종류의시금치노균병방제효과는모두 20% 이하로식물정유처리보다낮게나타났다. 난황유 0.3% 와 0.5% 농도에서 1 주일간격 2 회처리구에서병원균억제율이각각 84.5% 와 91.5% 로억제되어가장효과가우수한것으로나타났다. 검색어 시금치, 식물정유, 길항세균, 난황유, 노균병 ABSTRACT In this study, we evaluated environmental-friendly disease control methods using essential oils, antagonistic bacteria, or cooking oil and egg yolk mixture (COY). Among the five tested plant essential oils, Eucalyptus approximans oil shown the highest effect to control of the downy mildew disease (60% disease control value). Interestingly enough, well known antagonistic bacteria against various fungal pathogens were appeared lower control efficiency than plant essential oils. In field conditions, the cooking oil and yolk mixture (COY) treatments shown significantly decreased the downy mildew incidence with 0.3% (disease conttrol value 84%) and 0.5% concentration (disease control value 91%). Our findings suggest that the COY has a great potential as the eco-friendly downy mildew disease control formulation. Key words - Spinach, Essential oil, Antagonistic bacteria, Cooking oil and egg yolk mixture, Downy mildew *Corresponding author: Dong-Won Bae Tel: Fax: bdwon@gnu.ac.kr

2 28 Journal of Agriculture & Life Science 46(2) Ⅰ. 서론시금치 (Spinacia olerecea L.) 는명아주과에속하 는 1년생저온성식물로고대페르시아가원산지로아랍상인들에의해인디아와중국으로전해져현재까지식용으로이용되는작물이다 (Victor, 1949). 국내에서는주로나물로이용되고있으며미네랄, 비타민 B 복합체, 아스코르브산카로테노이드 (β -carotene, lutein, zeaxanthine) 플라보노이드, apocyanin 및 p-coumaric acid 등을함유하고있는좋은영양식품이다 (Zennie et al., 1977; Gerster 1993; Bergman et al., 2001). 또한시금치가포함하고있는비타민 E, 비타민 C, 셀레늄, 페놀산화합물, 카로테노이드및플라보노이드는산화방지효과가높은것으로알려져있다 (Chandha, 1996). 시금치노균병은전세계적으로발생하며경제적으로도중요한병이다 (Correll, et al., 1994). 노지재배보다시설재배에더큰피해를발생시킨다고알려져있고농가에서는버짐병으로불리기도하며, 시금치재배에서가장직접적인피해를입히는병으로잎에황색의병반을형성한다. 시금치노균병균 (Peronospora spinaciae) 은크로미스타계 (Chromista) 난균문 (Oomycota) 에속하는절대기생균으로살아있는기주에만기생한다. 또한전염원인난포자는토양중에서 2-3년간생존하는것으로알려져있으며 (Smith, 1988), 어린유묘부터수확까지전생육기에걸쳐서발생한다. 노균병발생은주로잎을이용하는시금치의품질저하와수확량감소의원인이된다. 이에따라노균병의발생을줄이기위해서는농약을사용하는화학적방제방법이있지만시금치는주로데쳐서나물로무쳐먹거나생으로먹는농산물이기때문에농약성분잔류되어있으면인체에농약성분이그대로노출될수있다. 또한실제해마다실시되는농산물품질관리원의조사에따르면시금치의잔류농약의검출율이제일높은것으로조사되었다. 따라서시금치노균병의친환경적방제법개발은유기농시금치를생산하는데필수적이며시금치의품질개선과농가소득증대및이미지제고에매우중요한요소가될것이다. 본연구는시금치의대량생산에문제가되는노균 병을친환경적으로방제하기위하여식물정유 (5 종 ), 난황유및길항세균 (3 종 ) 을처리하여노균병방제효 과를검정한결과를보고하고자한다. Ⅱ. 재료및방법 2.1 전염원수집 공시균주는경남남해군서면일대의대규모시 금치재배지에서시금치재배기간중에육안으로 조사하여노균병이대발생한재배지 (Fig. 1A) 에서 식물을수집한후 (Fig. 1B), 현미경으로검경하여 노균병균 (Peronospora spinaciae) 유주자낭를확인 한다음 (Fig. 1C), 노균병발병을위한전염원으로 사용하였다. 2.2 시금치재배환경 시금치품종은사계절시금치 (( 주 ) 해성씨드플러 스 ) 를사용하여노균병발병을위한시험포를조 성하였으며, 재배조건은 80 cm 의평이랑에 cm 간격으로줄파종하여하였다. 시험구의배치는난괴법 3 반복으로, 처리구의면적은 10 m 2 로사양토에서실시하였다. 실험중다른요소들 이관여치않도록비가림재배를하였다. 2.2 식물정유, 길항균및난황유처리 시금치노균병의방제를위한식물정유는시판되 는것 10 종과남부산림연구소에서 Eucalyptus 속식 물정유 10 종을분양받아실험에사용하였다. 인공적으 로배양이가능한잿빛곰팡이병균 (Botrytis cinerea) 과 탄저병균 (Colletotrichum gloeosporioides) 을이용하 여균사생장억제능력이큰것을 1 차선발하여 (data not shown) 최종적으로육계나무, 박하, 정향, Eucalyptus bicostata, E. approximans 를실험에 사용하였다 (Table 1). 길항균선발은토양을채취하여희석평판배양법 을이용하여단일클론을분리하여잿빛곰팡이병균 과대치배양을통하여길항력을확인하였다 (data not shown). 길항력이있는균주중에서잿빛곰팡 이병균의생장억제능력이우수한것을선발하여 MIDI gas chromatography(sherlock, USA) 를이용 하여동정하였다. 균주는일반적으로병원균에길항

3 Lee et al. : Control of Spinach Downy Mildew Using Essential Oil, Antagonistic Bacteria, Cooking Oil and Egg Yolk Mixture 29 능력을가지는것으로알려져있는 Bacillus subtilis, Paenibacillus polymyxa, Pseudomonas fluorescens 로동정되었으며분리된균주를배양하여실험에사용하였다. 난황유제조방법은 Jee et al.(2005) 이개발한방 법을응용하여제조하였다. 제조방법은계란노른자 (1 개 ), 물 (50ml) 및식용유 ( 올리브유, 100ml) 를믹서기를이용하여난황유유상액을제조한후, 물에잘확산되는것을확인한후대량의물에농도별로희석하여방제시험에공시하였다. Fig. 1. Downy mildew disease of spinach. (A) symptom of the downy mildew disease in a commercial field, (B) leaf symptom of downy mildew and (C) sporangium of the pathogen, Peronospora spinaciae. Table 1. List of plant essential oils Essential oil * Scientific name Extract from plant part Cinnamon * Cinnamomum zelanicum Bark Hyssop * Hyssopus officinnalis Leaf Clove bud * Syzigium aromaticum Bud Eucalyptus Eucalyptus bicostata Leaf Eucalyptus Eucalyptus approximans Leaf *Essential oils were purchased from Herb Mall Co. Ltd. (Seoul, Korea). 2.3 처리및방제가산출 시금치노균병을발생시키기위해서파종후 일경에시금치가 10 엽이상에서 1 엽당크기 가 10 cm 이상되었을때수집한전염원을약 20 cm 2 면적에약 50 g 전염원을시금치밭에뿌리 고, 물뿌리개를이용하여충분한수분이유지되도 록하여발병을되도록조장하였다. 전체시금치 포장에서노균병이약 30% 정도발병하고각 시금치잎의병반면적율이 5% 정도일때선발된 식물정유와길항미생물및난황유를처리하여 방제가를산출하였다. 방제가산출은노균병재 발생율 ( 방제된면적에대한재발생면적율 ) 을조 사하였다. 식물정유의처리방법은각각의식물정유 10 ml 를 DMSO(dimethyl sulfoxide) 10 ml 에녹이고 녹인용액을다시최종부피가 1 L 가되게물에 희석하여사용하였다. 대조구로는 10 ml 의 DMSO 를 1 L 의물에희석한것을사용하였다. 길항미 생물의처리방법은 PDB 배지에서대량으로배양 하고, 처리시배지부수효과를줄이기위해세균의배양액을원심분리하여멸균수에세척하고 10 6 개 /ml 농도로다시희석하였다. 희석액을평당약

4 30 Journal of Agriculture & Life Science 46(2) 1 L 정도를노균병이발생한시금치포장에분무기를이용하여잎의앞면과뒷면에고루살포하였다. 난황유의처리방법은난황유가만들어지는식용유의농도가각각 0.1, 0.3, 0.5와 1% 가되게하고노균병발생포장에처리하였다. 2.4 통계분석식물정유, 길항세균및난황유의방제가조사는 SAS(SAS Institute, Inc., 1989, Cary, NC) program 을이용하여 ANOVA 분석을하였으며, 처리간의비교를위하여 Turkey's test (p = 0.05) 를실시하였다. Fig. 2. The effects of the plant oils to control the downy mildew disease. The oils were applied only once. Different letters show statistically significant differences among treatments according to the Tukey HSD (p=0.05). Ⅲ. 결과및고찰 3.1 식물정유의노균병방제효과시판되는식물정유 ( 육계나무, 박하, 정향 ) 3종과 Eucalyptus 속식물정유 (E. bicostata, E. approximans) 2종의시금치노균병방제효과를조사한결과 1회처리구의발병억제율은시판되는육계나무, 박하와정향오일이각각 34.0, 45.5, 20.5% 였으며, Eucalyptus 속오일이 E. bicostata, E. approximans 각각 52.5, 60.0% 로 E. approximans 오일의억제율이가장높은것으로나타났다 (Fig. 2). 반면 Clove bud 오일의경우잿빛곰팡이나탄저병에억제효과가있는것으로나타났으나시금치노균병에대한억제효과는가장낮은것으로나타났다 (Data not shown). 식물정유를 1주일간격 2회처리구의노균병발생억제율은 1회처리와비교하였을때시판되는식물정유의경우 Cinnamon(34.0% 에서 46.0% 로 ) Hyssop(45.5% 에서 50.5% 로 ), Clove bud(20.5% 에서 51.5% 로 ) 모두노균병억제율이높아지는것으로나타났다. Eucalyptus 속오일의경우 E. bicostata (52.5% 에서 46.0% 로 ), E. approximan (60% 에서 60% 로 ) 노균병억제효과가떨어지거나변화가없는것으로나타났다. 특히 Clove bud 오일의경우 20% 에서 51% 로억제율이 31% 증가하여다른 4종의오일보다월등히 2회처리효과증가가높은것으로나타났다 (Fig. 3). Fig. 3. Inhibitory effects of the disease incidence by applied the plant oils. The oils were applied two time with a week interval. Different letters show statistically significant differences among treatments according to the Tukey HSD (p=0.05). 육계나무, 박하, 정향및 Eucalyptus 속오일은 본연구에서나타난시금치노균병이외에공기전 염성병해인 Botrytis cinerea(siripornvisal et al., 2009), 토양점염성병균인 Rhizoctonia solani (Seema & Devaki, 2010) 심지어는식중독균인 Listeria monocytogenes(cava et al., 2007) 의생 장도억제한다고보고되어있다. 각균마다억제효 과는다르나본실험에서오일처리효과는균의생 물학적특성이나처리환경에따라오일처리효과가 달라질수있는것이라판단되었고, 오일처리최적 조건은기온이나습도및온도를고려하여처리하 여야최적의방제효과가나타날수있을것이라판 단된다.

5 Lee et al. : Control of Spinach Downy Mildew Using Essential Oil, Antagonistic Bacteria, Cooking Oil and Egg Yolk Mixture 선발된길항균의노균병방제효과토양에서선발된길항균의시금치노균병방제효과는식물정유보다대부분억제율이낮은것으로나타났다. 1회처리구의노균병발생억제율은 Bacillus subtilis 가 17.5%, Paenibacillus polymyxa 가 7.5%, Pseudomonas fluorescens 가 3.0% 로나타났다 (Fig. 4). 1주일간격 2회처리구에서는 B. subtilis 가 22.5%, P. polymyxa 가 9.0%, P. fluorescens 가 4.5% 로각각 5.0%, 1.5%, 1.5% 억제율이증가하였다. 식물정유 2회처리결과에서억제효과가떨어지거나변화가없는것으로나타난반면선발된길항균은모두억제율이증가하는것으로나타났다 (Fig. 5). B. subtilis 와 P. polymyxa 의경우광범위한펩티드대사체 (peptid metabolites) 를생성하는데이는항세균활성및항진균활성이있다고보고되어있다 (Leifert et al., 1995; Walker et al., 1998; Beatty & Jensen, 2002; Gong et al., 2006). 또한 B. subtilis 에는항진균활성물질인 alboleutin, bacitracin, botrycidin, clorotetain, fengycin, iturins 및 ihizocticins 를생산한다고보고되어있다 (Chitarra et al., 2003). 본실험에서도출된결과는식물정유의경우병원균의살균효과가지속되지않는길항미생물의경우억제효과가지속되는것으로판단되며, 이를꾸준히사용했을때화학적방제를하지않는유기농시금치재배에유용할것으로판단된다. 본연구에서분리되어시험에사용된 B. subtilis, P. polymyxa, 및 P. fluorescens 이외에도차후더많은길항능력을가진세균및곰팡이가분리되어야하며친환경시금치재배를위하여적용되어야할것으로사료된다. Fig. 4. Suppression of Downy mildew disease by the selected antagonistic bacteria. The bacteria were treated one time. Different letters show statistically significant differences among treatments according to the Tukey HSD (p = 0.05). Fig. 5. Effect of the antagonistic bacteria treatments to reduce the disease incidence. The bacteria were treated twice with a week interval. Different letters show statistically significant differences among treatments according to the Tukey HSD (p = 0.05). 3.3 난황유의노균병방제효과난황유농도별시금치노균병방제효과는선발된길항균처리나식물정유처리보다월등히방제효과가높은것으로나타났다. 1회처리구에서노균병방제효과는 0.1% 농도에서 20.5% 의억제율을보여방제효과가낮은것으로나타났지만 0.3% 와 0.5% 의농도에서는억제율이각각 63.0% 와 88.5% 로방제효과가높은것으로나타났다 (Fig. 6). 또한 1주일간격 2 회처리구에서는 1회처리보다높은방제효과가증가하여 0.1% 농도에서 2회처리하였을때 1회처리보다 12.5% 높은 33% 의억제율을보였고 0.3% 와 0.5% 에서는각각 23.5% 와 3% 가증가한 84.5% 와 91.5% 의높은억제율을보였다 (Fig. 7). 국내에서난황유의식물병방제효과대한기록은흰가루병에대하여가지과작물에파프리카, 가지, 토마토 (Kwon et al., 2008; Lee et al., 2008), 엽채소류에상추 (Jee et al., 2008), 박과작물에오이 (Jee et al., 2005), 화훼류에장미 (Jee et al., 2008) 등이보고되어있다. 본연구와같이기주식물은다르지만오이에발생하는노균병방제에효과가있다는보고도있다 (Jee et al., 2009). 본연구에서는난황유의시금치노균병방제효과가우수한반면

6 32 Journal of Agriculture & Life Science 46(2) 않는것이안전하다는유의사항 (Jee et al., 2008) 을지켜난황유를사용하면친환경제제로노균병방제에활용할수있을것으로판단된다. 특히시금치는거의땅가에붙어자라는식물이기때문에난황유살포시시금치의잎표면에고르게분사해야방제효과가더높게나타나므로난황유살포시주의가요구된다. Fig. 6. The downy mildew disease were reduced by cooking oil and egg yolk mixtures (COY). the COYs were spread one time. Different letters show statistically significant differences among treatments according to the Tukey HSD (p = 0.05). Ⅳ. 감사의글 본연구는농림수산식품부지역농업클러스터활성화지원사업 ( 사업명보물섬남해시금치명품클러스터 ) 의지원에의해이루어진것임. Literature Cited Fig. 7. The disease incidences were reduced by COY treatments. The COYs were applied twice with a week interval. Different letters show statistically significant differences among treatments according to the Tukey HSD (p = 0.05). 약해도발생하였는데난황유 1% 농도를처리한결과 시금치의생육을저해하고잎의색을갈변시키는 현상이나타나 1% 난황유는친환경제제로적절치못 한결과가나타났다. 농촌진흥청에서발표한난황유 사용법의주의사항인작물별, 재배유형별, 생육시기 별로난황유에대한반응 ( 생육억제및약해발생 ) 이 다를수있으므로예비시험후사용하고특히시금 치는저온에서도생육이가능한작물이나동절기생 장억제혹은잎에반점발생등의장애를유발시킬 우려가있으므로하우스내최저온도가 5 이하및 최고온도 35 이상인저온기와고온기에는사용하지 Beatty P. H. and S. E. Jensen Paenibacillus polymyxa produces fusaricidin-type antifungal antibiotics active against Leptosphaeria maculans, the causative agent of blackleg disease of canola. Can. J. Microbiol. 48: Bergman, M., L. Varshavsky, H. E. Gottlieb, and S. Grossman, The antioxidant activity of aqueous spinach extract: chemical identification of active fractions. Phytochemistry. 58: Cava, R., E. Nowak, A. Taboada and F. Marin-Iniesta Antimicrobial Activity of Clove and Cinnamon Essential Oils against Listeria monocytogenes in Pasteurized Milk. J. Food Prot. 70: Chandha, S. L Natural sources of antioxidants and their adequacy in diet to prevent atherosclerosis. Mediquest. 143: Chitarra, G. S., P. Breeuwer, M. J. R. Nout, A. C. van Aelst, F. M. Rombouts and T. Abee An antifungal compound produced by Bacillus subtilis YM inhibits germination of Penicillium roqueforti conidiospores. J. Appl. Microbiol. 94: Correll, J. C., T. E., Morelock, M. C., Black, S. T., Koike, L. P., Brandenberger, and F. J. Dainello

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212 Research in Plant Disease Vol. 20 No. 3., 7 (Kim, 2004). Colletotrichum (Park Kim, 1992),.. (Agrios, 2008)... 1,202, 256 92, 147, 11 (RDA, 2014).

212 Research in Plant Disease Vol. 20 No. 3., 7 (Kim, 2004). Colletotrichum (Park Kim, 1992),.. (Agrios, 2008)... 1,202, 256 92, 147, 11 (RDA, 2014). 식물병연구 Research Article Open Access Res. Plant Dis. 20(3) : 211-215(2014) http://dx.doi.org/10.5423/rpd.2014.20.3.211 Protective Activity of the Mixtures of Pine Oil and Copper Hydroxide against Bacterial