05_06_HM _rev8_ hwp

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1 Korean Journal of Microbiology (2014) Vol. 50, No. 3, pp DOI Copyright c 2014, The Microbiological Society of Korea 보문 Bacillus sp. CS-52 를이용한고추탄저병 (Colletotrichum gloeosporioides) 방제특성 권정자 1,2 이중복 1 김범수 1 이은호 1 강경묵 1 심장섭 2 주우홍 3 전춘표 4 권기석 1 * 1 안동대학교생명자원과학과, 2 청송군농업기술센터, 3 창원대학교생물학과, 4 안동과학대학교의약품질분석과 Biological Control of Anthracnose (Colletotrichum gloeosporioides) in Red Pepper by Bacillus sp. CS-52 Joung-Ja Kwon 1,2, Jung-Bok Lee 1, Beam-Soo Kim 1, Eun-Ho Lee 1, Kyeong-Muk Kang 1, Jang-Sub Shim 2, Woo-Hong Joo 3, Chun-Pyo Jeon 4, and Gi-Seok Kwon 1 * 1 Department of Bioresource Sciences, Andong National University, Andong , Republic of Korea 2 Cheongsong Agricultural Technology Center, Gyeongbuk , Republic of Korea 3 Department of Biology, Changwon National University, Kyungnam , Republic of Korea 4 Department of Medicine Quality Analysis, Andong Science College, Andong , Republic of Korea (Received May 27, 2014 / Accepted August 4, 2014) This study was carried out in order to develop a biological control of anthracnose of red pepper caused by fungal pathogens. In particular, this study focuses on the Colletotrichum species, which includes important fungal pathogens causing a great deal of damage to red pepper. Antagonistic bacteria were isolated from the soil of pepper fields, which were then tested for biocontrol activity against the Colletotrichum gloeosporioides anthracnose pathogen of pepper. Based on the 16S rrna sequence analysis, the isolated bacterial strain CS-52 was identical to Bacillus sp. The culture broth of Bacillus sp. CS-52 had antifungal activity toward the hyphae and spores of C. gloeosporioides. Moreover, the substances with antifungal activity were optimized when Bacillus sp. CS-52 was grown aerobically in a medium composed of 0.5% glucose, 0.7% K 2HPO 4, 0.2% KH 2PO 4, 0.3% NH 4NO 3, 0.01% MnSO 4 7H 2O, and 0.15% yeast extract at 30. The inhibition of spore formation resulting from cellulase, siderophores, and indole-3-acetic acid (IAA), were produced at 24 h, 48 h, and 72 h, respectively. Bacillus sp. CS-52 also exhibited its potent fungicidal activity against anthracnose in an in vivo test, at a level of 70% when compared to chemical fungicides. These results identified substances with antifungal activity produced by Bacillus sp. CS-52 for the biological control of major plant pathogens in red pepper. Further studies will investigate the synergistic effect promoting better growth and antifungal activity by the formulation of substances with antifungal activity. Keywords: Bacillus sp. CS-52, Colletotrichum gloeosporioides, antagonistic microorganism, antifungal activity, red pepper anthracnose 한국에서고추는시설재배로연중생산되고있으며, 노지에서는 5월중하순부터재배하여가을에수확한다. 최근매년전국의고추생산량이감소하고있는추세이며, 특히 2007년이후재배면적당생산량이계속해서줄어들고있다. 그이유는병해로인한수량의감소로매년강수량과바람등의기후변화에따라탄저병과역병으로인하여크게발병할경우는탄저병에의해 10%-80%, 역병에의해 100% 까지수량이감소할수있다고보 These authors contributed equally to this work. *For correspondence. gskwon@andong.ac.kr; Tel.: ; Fax: 고되었다 (Badadoost, 2009). 국내에서고추에발생하는병해는 26종이며, 고추의주요생산제한요인중하나인탄저병은오래전부터알려진주요병으로우리나라뿐만아니라전세계적으로많은작물에큰피해를주고있다 (Okayama and Tsujimeto, 1994). 고추탄저병균간의분류학적특성은분생포자형태, 강모 (Seta), 균총색깔, 균사생장과병원성의특징에따라 Colletotrichum gloeosporioides, C. acutatum, C. coccodes, C. dematium과 Glomerella cingulata로서모두 5 종이분리동정되어국내에서보고되고있다 (Park and Kim, 1992). 특히, 경북북부지역에서발병하는고추탄저병균은 C. gloeosporioides, C. acutatum과 C. coccodes로 3개종이보고되었으며, 그중에

2 202 Kwon et al. 서도 C. gloeosporioides가우점하는종으로보고되고있다 (Kwon and Lee, 2002; Im and Lee, 2004). C. gloeosporioides는청고추와홍고추에대한병원성과분생포자형태의배양적특성에따라 G와 R계통으로분리하고있다. G계통은청고추와홍고추에서탄저병을일으키고, PDA 배지상에분생포자를형성하며, 자낭반과강모 (Seta) 는형성하지않고, 분생포자는한쪽끝이좁거나둥글고, 균총의색깔은흰색에서회색빛을형성한다. 또한, R 계통은붉은고추에서병징을나타내며, PDA 배지에서분생포자와자낭반을형성하고양끝이둥글고무딘분생포자를형성하며균총의색깔은회색내지어두운색을나타낸다고보고되었다 (Farley, 1967; Kim et al., 1986). 현재까지국내에서진행된고추탄저병에대한연구는주로살균제및병원성에대한고추의저항성에관한것으로대부분이병원균에대한길항성을나타내는미생물을이용하여방제가확인하는실정이다. 길항미생물을이용하는생물학적방제는세포벽을분해하는용균작용을보이는 Bacillus sp. (Kim and Kim, 1997; Kim et al., 1997; Woo et al., 2007), Penicillium sp. (Imamura et al., 2000), Pseudomonas sp. (Jung and Kim, 2004) 와 Streptomyces sp. (Lee et al., 1990; Jeong et al., 2004) 등이생산하는항생물질에의해직접식물병원균의생육을저해하는항생작용과식물병원균에기생하면서식물에대한진균의병원성을억제하는기생작용이있다 (Lee et al., 2004). 또한, 생육공간에서영양분과같은생육에필요한인자를경쟁함으로써병원균의생육및증식을억제하는경쟁적길항작용 (Scher and Baker, 1982; Neilands, 1984; Paulitz and Loper, 1991; Ping and Boland, 2004; Jung et al., 2006) 과미생물이생산하는 exopolysaccharide (EPS), lipopolysaccharide (LPS), salicylic acid (SA), hydrogen cyanide (HCN), 2,3-butanediol 등의물질들에의해서식물의면역기능을활성화하여병에대한저항성을유도하는유도저항성작용등을들수있다 (Liu et al., 1995; Lee, 1997; Ping and Boland, 2004). 이와같이토양내미생물들의상호작용에의한생물학적방제기작을활용하여현재까지종합적방제법의일환으로활발히연구가수행되어오고있다 (Cook, 1990; Lee et al., 1990, 1999; Handelsman and Stabb, 1996). 본연구에서는경북북부청송지역에서발생빈도가높은고추탄저병균주중 C. gloeosporioides에대하여강한항진균활성을나타내는균주를분리하여, 길항균주가생산하는항진균물질의생산조건을검토하고고추탄저병을방제할수있는미생물제제의개발을위한가능성을조사하였다. 재료및방법길항균주의분리및선발다양한고추재배지역으로부터채취한토양균원시료로부터 10 g을멸균수 90 ml에넣어 30 에서 24시간진탕배양후 LB (Luria Bertani, Difco, USA) agar 배지에단계희석법으로도말하여 30 에서 3일간배양한후증식한균주들을선별분리하였다. 선별분리된균주들을순수분리한후, 1차스크리닝으로고추탄저병을방제할수있는균주를선발하기위해 PDA (Potato Dextrose Agar, Difco) plate 에서대치배양을하여, C. gloeosporioides 의생육을저해하는균주들을일차적으로선발하였다. 고추탄저병병원균주인 C. gloeosporioides는농촌진흥청국립농업과학원농업유전자원센터 (Korea Agricultural Culture Collection, KACC) 로부터 C. gloeosporioides KACC 분양받아사용하였다. 1차선별된균주로부터고추탄저병저해활성이우수한균주를선발하기위해 LB 액체배지에선발균주를접종한뒤 48시간, 30 에진탕배양한후, 배양액을 8,000 rpm, 15분간원심분리하여, 배양상등액을이용하여 paper disc diffusion 방법으로항진균활성을보이는균주중활성이우수한균주를최종적으로분리하여본연구에사용하였다. 길항세균배양학적특성및생장특성최종선발된길항균의배양시간에따른생장및항진균활성물질의생산특성을조사하기위하여 Davis 최소배지 [0.5% glucose, 0.7% K 2HPO 4, 0.2% KH 2PO 4, 0.1% (NH 4) 2SO 4, 0.01% MgSO 4 7H 2O, 0.01% yeast extract] 에균주를접종하고 30, 120 rpm 으로진탕배양하여생육활성은 600 nm에서흡광도를측정하여균주의배양학적특성을조사하였다. 길항세균의동정최종선발된항진균활성을나타내는균주의정확한분류학적동정을위하여주사전자현미경검경을통해형태학적인특성과 16S rrna 유전자염기서열을통하여계통분류를확인하였다. 길항세균의항진균성활성물질의생산길항균이생산하는항진균성활성물질에의한고추탄저병균생육의길항정도를측정하기위해먼저길항세균을 Davis 최소배지에 30 에서 2일간전배양한후 8,000 rpm에서 15분간원심분리한후, paper disc diffusion method를이용하여 conidium/ml로 C. gloeosporioides가접종되어진 PDA plate에상등액 100 μl를점적한 paper disc (Ø8 mm) 를올려, 28 에서 5일간배양하여항진균활성을조사하였다. 항진균성활성물질생산에대한탄소원, 질소원및무기염류의영향탄소원의첨가에따른항진균성활성물질의생산에대한영향을조사하기위하여 Davis 최소배지에탄소원으로 glucose를제외시키고 7종의탄소원인 mannose, glucose, fructose, sucrose, lactose, maltose와 starch를각각 0.5% 씩첨가하여탄소원에따르는최적화를통해항진균활성을조사하였다. 또한최적배지조성선택에있어항균활성이가장우수한탄소원을선택하여 0.5%, 1.0%, 1.5% 와 2.0% 를각각첨가하여항균활성이가장높은탄소원농도를선정하여최적배지의조성농도로설정하였다. 질소원에따르는최적화를위해 Davis 최소배지에서 (NH 4) 2SO 4 를제외시키고, 13종의질소원인 (NH 4) 2SO 4, KNO 3, NH 4HCO 3, NaNO 3, NH 4NO 3, NH 4Cl, (NH 4) 2PO 4, peptone, tryptone, malt extract, soytone, yeast extract, casamino acid를각각 0.1% 첨가하여항균활성을조사하였다. 최적배지조건선택에있어항균

3 고추탄저병생물학적방제특성 203 활성이가장우수한질소원을선택하여 0.1%, 0.2%, 0.3%, 0.4% 와 0.5% 를각각첨가하여항균활성이가장높은질소원농도를선정하여최적배지의조성농도로설정하였다. 무기염의경우 MgSO 4 7H 2O를제외시키고 CoCl 2, FeCl 2, CaCl 2, MgSO 4, MnSO 4, KCl를각각 0.01% 첨가하여각각에대한항진균성활성물질생산에미치는영향을조사하였다. 또한최적배지조건선택에있어항균활성이가장우수한무기염을선택하여 0.01%, 0.05%, 0.10%, 0.15% 와 0.2% 를각각첨가하여항균활성이가장높은무기염농도를선정하여최적배지의조성농도로설정하였다. 항진균활성은선발된균을각각의배지조건에서 30, 3일간 120 rpm으로진탕배양하여배양액을 8,000 rpm에서 15분간원심분리한후상등액을이용하여항진균활성능을조사하였다. 길항세균배양액의 ph 및온도에대한안정성최종선발된길항세균배양액의 ph에대한안정성은최적화된배지에분리균주를 1% (v/v) 접종한후, 30 에서 2일간 120 rpm으로진탕배양하여상등액을 ph 3, 5, 7, 9 그리고 11로조정하여 ph 변화에따른항진균활성을조사하였다. 온도에대한안정성은상등액을온도 -20, -4, 0, 4, 10, 20, 30, 40, 50, 70 그리고 90 에서 15분동안처리하고, 110과 121 에서 15분간고압열처리하여각각의온도에따른안정성을조사하였다. 탄저병균에대한포자발아저해능길항균주의탄저병원균에대한포자발아저해능을조사하기위하여 C. gloeosporioides 포자현탁액 ( conidium/ml) 과길항균주배양상등액을 50배희석하여 1:1의비율 ( 최종 100배희석 ) 로 96-well plate에함께분주한후 24시간동안배양한다음배양된병원균의생장정도를흡광도 (660 nm) 로측정하였으며, 저해율은다음과같이나타내었다 (Wedge et al., 2001). 저해율 (%) = [100 - ( 처리구흡광도 / 대조구흡광도 )] 100 항진균활성물질인 siderophore, cellulase 와 indole-3-acetic acid (IAA) 생성능길항균주의 siderophore의생산여부측정은 CAS (chrome azurol S) blue agar plate assay에의하여측정하였다. 길항균주를접종하고 30 에서 3일간배양하면서, 시간변화에따라나타나는 colony 주위에 orange halo zone의생성으로조사하였다. 생산된 siderophore의측정은 CAS liquid assay에의하여배양된길항균주상등액에 CAS assay solution를첨가하여흡광도 630 nm에서측정하여 siderophore의활성을조사하였다 (Schwyn and Neilands, 1987; Crosa, 1989; Bergeron and McManis, 1991). Cellulase의생산능측정은 1% CMC (Carboxymethyl cellulose, Junsei, Japen) 를첨가한배지에길항균주를접종배양한후이를이용하여 DNS 방법으로조사하였다 (Miller, 1959). 대표적인식물생장호르몬인 IAA의생성능은 Tang과 Bonner 방법 (1947) 에의해확인하였고, IAA의생산량을알아보기위해 IAA의전구물질인 L-tryptophan을 0.1% 첨가한최소배지에선별된균주를접종하고 30 에서 3일간배양하였다. 배양시간에따라생성되는 IAA를조사하기위해, 배양액을 8,000 rpm에서 10분간원심분리하여상등액을 salkowski reagent (5% perchoric acid 100 ml, 0.05 M ferric chloride 2 ml) 와혼합하여 30분간반응시킨후반응액의색상이분홍색으로전환된것을 IAA 생성능이있는것으로결정하였다. 길항세균의탄저병에대한기내항진균실험길항세균의고추과실에대한항진균활성을검정하기위해습실용기를만들어기내검정을실시하였다. 직접제작한습실용기 (28 cm 23 cm 9 cm) 에고추과실을처리구별로용기에각각 10개씩 ( 청고추 5개, 홍고추 5개 ) 담고, 고추탄저병포자현탁액 ( conidium/ml) 을고추과실에가는바늘을이용해상처접종으로처리하였다. 고추표면의물기가마른다음젤라틴 1.5% 의희석수가첨가된길항세균배양액을 100배희석하여각각의시료에스프레이후뚜껑을닫고, 25 항온기에서 6일간관찰한후병반유무를확인하여나타나지않는열매의수를방제가로산출하였다. 병발병억제력 (%)= [( 길항미생물무처리구발병률- 길항미생물처리구발병률 )/ 길항미생물무처리구발병률 ] 100 결과및고찰길항균주의분리및선발고추탄저병에대해강한항진균활성을갖는길항세균을분리및선발을위해총 100여점의토양시료로부터균주를분리하였고, 분리균주중항진균활성을조사하여 1차적으로 5종의균주를선발하였다. 선발된 5종 (#31, #43, #52, #61, #72) 모두탄저병에대하여높은항진균활성을각각 8.8 mm, 7.4 mm, 9.7 mm, 4.4 mm와 7.1 mm로각각나타났으며, 이들중항진균력이가장높게나타난 #52을최종선발하여 CS-52로명명후실험에사용하였다. 길항세균의동정최종선발된길항세균의동정을위하여분리균주인 CS-52을 LB배지를이용하여 30 에서순수배양한후생화학적특성을조사한결과내생포자를형성하는 Gram-positive로세포크기는 μm으로짧은간균으로조사되었다. 16S rrna 유전자염기서열을이용한동정결과 1,382 bp의염기서열을얻어 NCBI (National Center for Biotechnology Information) 의 BLAST 를이용하여유전자상동성검색한결과를기반으로 Bacillus sp. 와 99% 의유연관계를보여 Bacillus sp. CS-52로명명하였으며다른균주들과의유연관계를 Fig. 1에나타내었다. 항진균활성물질의생산을위한영향및최적조건 Bacillus sp. CS-52가생산하는항진균물질의생산에미치는탄소원의영향은 Table 1과같다. 7종의탄소원중에서 glucose를

4 204 Kwon et al. Table 1. Effects of various sources, nitrogen sources and mineral salt sources on the antifungal activities from Bacillus sp. CS-52 Carbon sources Antifungal activity (Ø mm) Nitrogen sources Antifungal activity (Ø mm) Salt sources Antifungal activity (Ø mm) Control 0 Control 0 Control 0 Maltose 6.7 (NH 4) 2SO CoCl Mannose 6.4 KNO FeCl Lactose 6.2 (NH 4) 2PO CaCl 2 8 Glucose 7.85 NaNO KCl 9.5 Sucrose 6.5 NH 4NO MgSO Fructose 7.5 NH 4Cl 5.8 MnSO 4 12 Starch 5.4 NH 4HCO Yeast extract 9.8 Peptone 7.5 Tryptone 5.9 Soytone 7.49 Malt extract 6.0 Casamino acid 6.19 첨가하였을때가장양호한항진균활성을나타냈으며, 전반적으로탄소원에대해서는항진균활성이큰차이가없는것으로조사되었다. 그러나항생물질생산성과경제성을고려한다면대량배양시최적탄소원으로 7.85 mm의항진균활성을보인 glucose를최적탄소원으로사용하는것이적합할것으로보인다. 최적배지조성을위한 Glucose의 0.5%, 1%, 1.5% 와 2% 에서각각배양한결과 0.5% 에서 8.4 mm로가장높은활성을보였으며, 1%, 1.5% 와 2% glucose농도에서는 7.84 mm, 8.25 mm와 8.15 mm로각각조사되었다. 또한 Kim 등 (1997) 도항진균활성물질의생산성이 glucose에서가장우수하다고보고하였다. Davis 최소배지를기본으로하여질소원의영향에따른항진균활성을조사한결과 Table 1과같았다. 다양한질소원에서생육활성및항진균활성을보였으나, 특히유기질소원에서의존적인생육활성과항진균활성을보이는것으로조사되었다. 무기 (A) (B) Fig. 1. (A) Neighbor-joining tree based on almost-complete 16S rrna gene sequences, showing phylogenetic relationships between strain CS-52 and species from Bacillus genus. Numbers at nodes indicate levels of bootstrap support (%) based on 1,000 resampled datasets; only values above 70% are given. Bar, 0.1 substitutions per nucleotide position. (B) Scanning electron microscopic photograph of strain CS-52 ( 6,000).

5 고추탄저병생물학적방제특성 205 질소원에서는 NH 4NO 3 를첨가하였을때항균활성이 7.7 mm로나타났으며, NH 4NO 3 의최적배지조성에따르는농도에조사한결과항진균활성이 0.1%, 0.2%, 0.3%, 0.4% 와 0.5% 에서각각 7.7 mm, 7.8 mm, 9.0 mm, 7.8 mm와 6.0 mm로각각조사되어 0.3% 첨가시가장높은항진균활성을보이는것으로조사되었다. Davis 최소배지에서유기질소원을 yeast extract 0.01% 첨가시 9.8 mm로가장높은항진균활성을나타내었다. 최적배지농도조성을위해 yeast extract의첨가농도에따라조사한결과 0.01%, 0.05%, 0.1%, 0.15% 와 0.2% 에서 6.0 mm, 8.2 mm, 9.0 mm, 11.8 mm와 11.0 mm로각각조사되어 yeast extract 0.15% 를최적배지의유기질소원농도로설정하였다. 일반적으로길항세균의성장과항진균활성물질생산능이무기질소원보다유기질소원에서높게나타나는결과는 Bacillus sp. 이생산하는항생물질의다른보고들과유사한결과이다 (Kim et al., 1997; Jeong et al., 2002). 무기염에대한항진균활성물질의생산성을조사한결과 Bacillus sp. CS-52은 Table 1에서와같이 MnSO 4 를첨가하여배양하였을때항진균억제활성이 12 mm로가장높게나타났다. 그외 FeCl 2, CaCl 2, KCl과 MgSO 4 7H 2O에서도각각높은 항진균력을보였으며, 이러한결과로보아 Bacillus sp. CS-52을미생물제제화하여현장에이용한다면이미토양에잔재하고있는비료성분을이용하여높은항진균물질을생산하여병해방제효과가있을것으로사료된다. 이러한결과는 Jeong와 Kim (2003) 의보고에서 KCl, MgCl 2, NaCl, BaCl 2 첨가시항진균물질의생산이양호하다는결과와유사한것으로보인다. 최적배지조건에서 Bacillus sp. CS-52 의항진균활성 Bacillus sp. CS-52 균주가생산하는항진균물질의최적생산배지조성은 0.5% glucose, 0.7% K 2HPO 4, 0.2% KH 2PO 4, 0.3% NH 4NO 3, 0.01% MnSO 4 와 0.15% yeast extract로각각조사되었다. 이러한최적배지조건에서최적 ph와최적온도그리고배양시간에따른성장및항진균활성물질의생산특성을조사한결과 Fig. 2와같이 18-24시간사이에정체를보이는것으로나타났으나, 시간이지남에따라 2차증식을보였으며배양 36시간에가장높은 OD 600 값 1.5로조사되었으며, 최대항진균활성은지속적으로증가하다배양시간 60시간에 13.3 mm로최대치를보이다감소하는것으로조사되었다. 이러한결과로보아 Bacillus sp. CS-52은대수성장기를지나생산되는 2차대사산물에의해항진균활성을나타냄을추정할수있다. 이러한결과는 Hwang과 Kim (1995) 에의해보고된결과와유사하게나타났으며, 항진균성활성을나타내는길항세균은주로 2차대사산물을산출함으로써활성을나타냄을추정할수있다 (Suzi, 1992). Fig. 2. Antifungal activity of Bacillus sp. CS-52 culture broth against C. gloeosporioides according to cultivation time. Data are expressed as means ± SE, n=3. Bacillus sp. CS-52 균주배양액의 ph 및온도에대한안정성항진균활성을보이는 Bacillus sp. CS-52 배양액의 ph에대한안정성에대한실험결과 Fig. 3에서와같이배양액이고추탄저병에대한억제력이 ph 3-11까지넓은범위에서나타났다. 이러한결과는우리나라토양의특성인산성에서보다더안정한균주로항진균활성을유지할수있을것으로사료된다. 또한 Bacillus sp. CS-52 배양액의온도에대한안정성을조사한결과 Fig. 3에서같이 30 에서도가장높은안정성을보였으며, 저온과고온의넓은범위에서도매우안정적으로조사되었으며, 특히고압열처리한배양액에서도항균활성을보였다. Naclerio 등 (1993) 의보고 Fig. 3. Effects of Bacillus sp. CS-52 culture broth on antifungal activity in various range of ph and temperatures showing stability of the activity. Data are expressed as means ± SE, n=3.

6 206 Kwon et al. (A) (B) (C) Fig. 4. Bacillus sp. CS-52 culture broth of fungal spore germination inhibition activity, and production of siderophore and cellulase [Arrows: C. gloeosporioides spore, (A) Spore germination inhibition activity, (B) Confirmation of orange halo zone by Bacillus sp. CS-52 in selection media of CAS blue agar, (C) Confirmation of halo zone by congo-red staining of LB agar plates containing carboxymethyl-cellulose (CMC)]. 에의하면 Bacillus cereus에서생산된 cerein의경우 ph 3.0에서 12.0까지넓은범위에서안정하다고보고된바있다. 이러한결과로보아 Bacillus sp. CS-52 배양액을토양에살포하였을때여름철과겨울철온도변화에따라토양내에서도안정적으로사료되어현장에서고추탄저병방제효과가우수할것으로사료된다. 탄저병균에대한포자발아저해및항진균활성물질 siderophore, cellulase 와 indole-3-acetic acid (IAA) 생성능길항세균 Bacillus sp. CS-52가생산하는항진균활성물질이고추탄저병원균의포자발아억제능조사결과, Fig. 4A와 5에서와같이최대생육기 48시간이후탄저병원균인 C. gloeosporioides 에대한포자발아억제율 72% 로가장높게나타났으며, 최종배양시간까지높은활성을유지하였다. 또한 Bacillus sp. CS-52가 CAS agar plate에서 orange halo zone을형성하여 siderophore 생산균주임을확인하였다 (Fig. 4B). 이러한물질은철이온특이결합물질로서식물병원균의생육을저해하는경쟁적길항작용을함과동시에식물이이용할수없는철을가용화시켜식물 성장에도움이된다. Bacillus sp. CS-52가생산하는 siderophore 의총생산량을조사결과 Fig. 5에서와같이최종배양 72시간까지지속적으로증가되는것으로조사되었다. Bacillus sp. CS-52 가높은항진균활성을나타내기위해서는길항물질과더불어 siderophore를최대생산할수있는조건에대해조사가필요할것으로사료된다. 항진균활성효소물질을조사하기위하여 CMC가첨가된 plate에 Bacillus sp. CS-52를 4일간배양후, congo red 시약을스프레이하여반응후투명환을조사한결과 Bacillus sp. CS-52 주위에넓게형성된투명환을통해 cellulase의생산을확인하였다 (Fig. 4C). 보다더정확한 cellulase의생산량을알아보기위해배양액을 12시간간격으로채취한뒤 DNS환원법으로측정한결과 Fig. 6에서와같이 Bacillus sp. CS-52의성장에따라 cellulase가생성됨을알수있었다. 이러한결과는고추탄저병뿐만아니라, 세포막이 cellulose와 β-glucan으로구성된고추역병에대해서도우수한방제효과가있을것으로사료된다. Bacillus sp. CS-52 균주배양액의식물생장촉진물질의생산을조사한결과 Fig. 6에서와같이배양시간 24시간일때 187 μg/mg IAA를생산하는것으로나타났으며, Bacillus sp. CS-52 균주는고추탄저병원균에대하여항균활성이우수하고식물뿌리의신장등작물의생육을촉진하는 PGPR (plant growth promoting Fig. 5. Production of siderophore and inhibition of spore germination by Bacillus sp. CS-52 according to cultivation time. Data are expressed as means ± SE, n=3. (Siderophore units: For A630 measurement, use the minimal medium as a blank, and use the minimal medium plus CAS assay solution as reference. The sample should have a lower reading than the reference. Siderophore units are defined as [(Ar As)/Ar] 100 = % siderophore units. Ar, Absorbance of the CAS solution plus the medium; As, Absorbance of the CAS solution plus the culture supernatant of the respective sample). Fig. 6. Production of cellulase and IAA by Bacillus sp. CS-52 according to cultivation time. Data are expressed as means ± SE, n=3 (Cellulase unit: One unit of cellulase defined as the amount of enzyme releasing 1 μmol of reducing sugar per min).

7 고추탄저병생물학적방제특성 207 Table 2. Disease control effect by Bacillus sp. CS-52 against C. gloeosporioides on red pepper at wetting treatment chamber Treatment Dilution Disease rate (%) Note A 0 B X Contamination C 30 test chamber D X1, A, Water treatment control B, Positive control, C. gloeosporioides treatment C, Bacillus sp. CS-52 culture broth with C. gloeosporioides treatment D, Pesticide (ace ) with C. gloeosporioides treatment rhizobacterium) 균주로식물생장촉진형생물방제제로서의활용이가능할것으로사료된다. Bacillus sp. CS-52 의탄저병에대한기내항진균활성처리구별습실용기를확인해본결과 Fig. 7에서물만처리한처리구에서는청고추, 홍고추모두가표면이깨끗하게병반이이병된흔적을찾아볼수없었으며, 반면고추탄저균처리구에서는모두가고추표면에이병되었음을관찰하였다. 길항미생물과탄저병원균처리구에서는청고추에서 3개가이병되었으며, 화학농약과탄저병원균처리구에서는홍고추 1개와청고추 4개가이병된것으로나타났다. 또한양성대조구인화학농약처리구의방제가는 50% 로조사되었으며, Bacillus sp. CS-52 배양액처리구에서 70% 으로화학농약처리구보다높게나타났다 (Table 2). 습실용기내에서탄저균에대한실험은항온기내에서온도및습실용기내에서의습도유지가병원균의이병조건에알맞은환경을제공되었으므로탄저균의활동성이활발해져탄저균실험에서다소방제력이차이가있었다고판단된다. 탄저병실험에서방제능력은 70% 정도로검정되었으며이는고추탄저병원균에대한미생물제제로서의산업적연구ㆍ개발가치가높은친환경농자재로활용가능한미생물자원이라보인다. 최근기후변화로인하여작물병해를일으키는병원균의변화와이를제어하기위한다양한화학농약및생물농약이지속적으로많은연구자들에의해보고ㆍ개발되어왔다. 하지만친환경농자재또는생물농약의경우산업화와현장적용을위한균주의대량생산을위한최적배지조성의완성을통해업체나지역농업기술센터에서쉽게배양하여사용하는것이중요하다. 본연구에서분리된 Bacillus sp. CS-52 균주를다양한탄소원, 질소원과무기염의종류에따라배지조성의최적농도를확립하여항균활성을조사하고, 이를기반으로지속적살포를통한현장에적용이가능성을보이나향후탄저병원균의종류에따른연구가필요할것으로사료된다. (A) (B) (C) (D) Fig. 7. Biological control effect by Bacillus sp. CS-52 culture broth against C. gloeosporioides on red pepper at wetting treatment chamber [Anthracnose disease was observed by C. gloeosporioides at 6 days, 25 after inoculation. Arrows: Susceptible reaction of C. gloeosporioides. (A) Negative control, water treatment, (B) Positive control, C. gloeosporioides treatment, (C) Bacillus sp. CS-52 broth with C. gloeosporioides treatment, (D) Pesticide(ace ) with C. gloeosporioides treatment]. Typical symptoms were developed on accessions of Capsicum spp. by pin-prick wound inoculation.

8 208 Kwon et al. 적요 본연구에서는고추재배지역의오염지토양으로부터탄저병원균인 Colletotrichum gloeosporioides에대하여항진균활성이우수한균주를분리하였다. 분리균주의생화학적특성을조사한결과내생포자를형성하는 Gram-positive이며, 세포크기는 2.5~ μm으로짧은간균으로 siderophore, cellulase와 IAA (Indole-3-acetic acid) 을생산하였다. 16S rrna 유전자염기서열분석결과 Bacillus sp. 와 99% 의상동성을나타내어 Bacillus sp. CS-52로명명하였다. Bacillus sp. CS-52의항진균활성물질을생산하기위한배양최적조건은 0.5% glucose, 0.7% K 2HPO 4, 0.2% KH 2PO 4, 0.3% NH 4NO 3, 0.01% MnSO 4 7H 2O, 0.15% yeast extract, ph 7과 30 로조사되었으며, 최적화된배양조건에서 36시간에최대성장을보이며, 고추탄저병원균에대하여 60시간배양조건에서가장높은 13.3 mm의항진균활성을보였다. 포자발아억제력은 48시간에가장높은억제력이보였고, siderophore는최종배양시간 72시간까지생성됨을확인하였다. 식물생장조절물질 IAA와활성효소인 cellulase의경우배양시간 24시간에최대생성됨을확인하였으며, C. gloeosporioides에대한실내에서의항진균활성검증결과화학농약보다더높은 70% 의방제가를나타내었다. 향후 Bacillus sp. CS-52 균주와배양액을이용하여생물학적친환경방제제로의제품화가가능할것으로사료된다. 감사의말 본연구는농림축산식품부생명산업기술개발 ( ) 에의해이루어진것이며, 이에감사드립니다. References Babadoost, M Phytopthora blight (Phytophthora capsici) of pepper and its management. The 2009 KSPP Fall Meeting and the 1 st Japan-Korea Joint Symposium. Bergeron, R.J. and McManis, J.S Synthesis of catecholamide and hydroxamate siderophore. In Winkelmann, G. (eds.), CRC Hanbook of Microbial Iron Chelates, pp CRC Press, Boca Raton, FL, USA. Cook, R.J Twenty-five years of progress towards biological. pp In Hornby, D. (eds.), Biological control of soil-borne plant pathogens, CAB International, Wallingford, UK. Crosa, J.H Genetics and molecular biology of siderophoremediated iron transport in bacteria. Micorbiol. Rev. 53, Farley, J.D Survival of Collectotrichum coccodes in soil. Phytopathology 66, Handelsman, J. and Stabb, E.V Biocontrol of soilborne plant pathogens. Plant Cell. 8, Hwang, B.K. and Kim, C.H Phytophthora blight of pepper and its control in Korea. Plant Dis. 79, Im, J.H. and Lee, D.G Red pepper anthracnose : Colletotrichum gloeosporioides, it's cultural variations and pathogenicity. Res Plant Dis. 10, Imamura, N., Ishikawa, T., Ohtuka, T., Yamamoto, K., Dekura, M., Fukami, H., and Nishida, R An antibiotic from Penicillium sp. covering the cocoon of the leaf-rolling moth, dactylioglyphatonica. Biosci. Biochem. 64, Jeong, H.G. and Kim, S.D Purification and characterization of an antifungal antibiotic from Bacillus megaterium KL 39, a biocontrol agent of red-papper phytophtora blight disease. Kor. J. Appl. Microbiol. Biotechnol. 31, Jeong, D.H., Park, K.D., Kim, S.H., Kim, K.R., Choi, S.W., Kim, J.T., Choi, K.H., and Kim, J.H Identification of Streptomyces sp. producing antibiotics against phytopathogenic fungi and its structure. J. Microbiol. Biotechnol. 14, Jeong, Y.K., Shin, M.J., Joo, W.H., and Choi, J.S Structural analysis of the antifungal antibiotic from Bacillus sp. YJ-63. Kor. J. Appl. Microbiol. Biotechnol. 30, Jung, H.K. and Kim, S.D Selection and antagonistic mechanism of Pseudomonas fluorescens 4059 against phytophthora blight disease. Kor. J. Microbiol. Biotechnol. 32, Jung, H.K., Kim, J.R., Woo, S.M., and Kim, S.D An auxin producing plant growth promoting rhizobacterium Bacillus subtilis AH18 which has siderophore-producing biocontrol activity. Kor. J. Microbiol. Biotechnol. 25, Kim, W.G., Cho, E.K., and Lee, E.J Two strains of Colletotrichum gloeosporioides Penz. causing anthracnose on pepper fruits. Korean J. Plant Pathol. 2, Kim, K.Y. and Kim, S.D Biological control of Pyricularia aryzae blast spot with the antibiotic substances produced by Bacillus sp. KL-3. Kor. J. Appl. Microbiol. Biotechnol. 25, Kim, Y.S., Son, J.K., Moon, D.C., and Kim, S.D Isolation and structure determination of antifungal antibiotics from Bacillus subtilis YB-70. Kor. J. Appl. Microbiol. Biotechnol. 25, Kwon, C.S. and Lee, S.G Occurrenceand ecological characteristics of red pepper anthracnose. Res. Plant Dis. 8, Lee, M.W Root colonization by beneficial Pseudomonas spp. and bioassay of suppression of Fusarium wilt of radish. Kor. J. Mycol. 25, Lee, Y.S., Choi, J.W., Kim, S.D., and Baik, H.S Isolation of antagonistic bacteria to Phytophthora capsici for biological control of phytophthora blight of Red Pepper. Kor. J. Life Science 9, 1 7. Lee, I.K., Kim, C.J., Kim, S.D., and Yoo, I.D Antifungal antibiotic against fruit rot disease of red pepper form Streptomyces parvullus. Kor. J. Appl. Microbiol. Biotechnol. 18, Lee, S.Y., Lee, S.B., Kim, Y.K., and Kim, H.G Effect of agrochemicals on mycelial growth and spore germination of a hyperparasite, Ampelomyces quisqualis for controlling cucumber powdery mildew. Kor. J. Pesti. Sci. 8, Liu, L., Kloepper, J.W., and Tuzun, S Induction of systemic resistance in cucumber by plant producing plant growth promoting rhizobacteria: duration of protection and effect of host resistance on protection and root colonization. Phytopathology 85, Miller, G.L Use of the dinitrosalicylic acid reagent for the determination of reducing sugars. Anal. Chem. 31, Naclerio, G., Ricca, E., Sacco, M., and de Felice, M Antimicrobial activity of a newly identified bacteriocin of Bacillus cereus. Appl. Environ. Microbiol. 59, Neilands, J.B Siderophores of bacteria and fungi. Microbiol. Sci. 1, Okayama, K. and Tsujimeto, A Occurrence of strawberry anthracnose caused by Glomerella cingulata (Stoneman) Spaulding et Schrenk and pathogenicity of the fungus. Ann. Phytopath. Soc. 60,

9 고추탄저병생물학적방제특성 209 Park, K.S. and Kim, C.H Identification, distribution and etiological characteristics of anthracnose fungi of red pepper in Korea. Korean J. Plant Pathol. 8, Paulitz, T.C. and Loper, J.E Lack of a role for fluoredcent siderophore production in the biological control of Phythium damping-off of cucumber by astrain of Pseudomonas putida. Phytopathology 81, Ping, L. and Boland, W Signals from the underground: bacrerial volatils promote growth in Arabidopsis. Trends Plant Sic. 9, Scher, F.M. and Baker, R Effect of Pseudomonas putida and a synthetic iron chelator on inductionof soil suppressiveness to Fusarium wilt pathogens. Phytopathology 72, Schwyn, B. and Neilands, J.B Universal chemical assay for the detection and determination of siderophores. Anal. Biochem. 160, Suzi, T Biological control of soil borne disease with antagonistic microbes. Prov. 92 agric BioTech Symp. On New Bio Pesticides Tang, Y.W. and Bonner, J The enzymatic inactivation of indole acetic acid I. Some characteristics of the enzyme contained in pea seedlinds. Arch. Biochem. 13, Wedge, D.E., Curry, K.J., Boudreaux, J., Pace, P., and Smith, B.J A microtiter assay demonstrates sensitivity and resistance profiles for Botrytis cinerea isolates from Louisiana strawberry farms. Adv. Strawberry Res. 20, Woo, S.M., Woo, J.U., and Kim, S.D Purification and characterization of the sidrophore from Bacillus licheniformis K11, a multi-functional plant growth promoting rhizobacterium. Kor. J. Microbial. Biotechnol. 35,