492 Korean Journal of Soil Science and Fertilizer Vol. 51, No. 4, 2018 Introduction 인삼은오갈피나무과에속하는다년생초본식물로서작물의특성상동일장소에서 3-5년동안장기간재배하므로작물의생육기간중기상, 재배환경및

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1 Korean J. Soil Sci. Fert. Vol.51, No.4, pp , 2018 Korean Journal of Soil Science and Fertilizer Article pissn : eissn : Suppressive Effect of Bacillus amyloliquefaciens KL87 on Alternaria Blight of Ginseng Ye-Ji Yoon, Jeong-Su Jeong, Jae-Hee Won, Jin-Ho Joo 1, and Kwang-Jae Lee* Ginseng & Medicinal Plants Research Institute, Gangwon ARES, Cheorwon 24054, Korea 1 Department of Biological Environment, Kangwon National University, Chuncheon 24341, Korea *Corresponding author: rsc@korea.kr A B S T R A C T Received: September 20, 2018 Revised: November 19, 2018 Accepted: November 28, 2018 Biological control of plant disease using microorganisms such as Bacillus, Trichoderma and Streptomyces is alternative method which can reduce disease in ginseng cultivation. Antagonistic microorganisms can replace chemical agent, and reduce yield losses caused by several pathogens. The purpose of this study was to develop eco-friendly microbial agents and application technologies for the safe production of ginseng through control of ginseng aerial disease. For the selection of antagonistic microorganisms to control ginseng aerial disease, soil samples were collected from ginseng cultivation field, and 260 kinds of bacterial microbes were isolated. Among the 260 isolated microbes, KL87 strain suppressing the growth of ginseng pathogenic bacteria, Alternaria panax and Botrytis cinerea, was selected. As a result of 16S ribosomal RNA sequence analysis, KL87 strain was identified as Bacillus amyloliquefaciens. Furthermore, biochemical characteristics of KL87 strain showed high identity (99%) with Bacillus amyloliquefaciens. In the ginseng cultivation field test, Bacillus amyloliquefaciens KL87 reduced the occurrence of ginseng Alternaria blight by 75.9%. These results strongly suggested that Bacillus amyloliquefaciens KL87 could be used as microbial agents for replacement of chemical agent. Keywords: Ginseng, Alternaria blight, Bacillus amyloliquefaciens, Alternaria panax. Suppressive effect of Bacillus amyloliquefaciens KL87 on Alternaria blight occurrence in ginseng cultivation field. C The Korean Society of Soil Science and Fertilizer. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non- Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

2 492 Korean Journal of Soil Science and Fertilizer Vol. 51, No. 4, 2018 Introduction 인삼은오갈피나무과에속하는다년생초본식물로서작물의특성상동일장소에서 3-5년동안장기간재배하므로작물의생육기간중기상, 재배환경및병충해등에의한피해를받기쉽다. 특히, 장기간의재배기간중점무늬병, 탄저병, 잿빛곰팡이병, 뿌리썩음병등의각종병해에의해매년 30-60% 의수량감소피해를입는것으로알려져있어, 이러한병해피해를효과적으로예방할수있는친환경농자재개발이필요한실정이다 (Kim et al., 2012). 인삼점무늬병은 Alternaria panax, Alternaria sp. 균에의해유발되어인삼의줄기, 잎또는열매등에흑갈색의부정형병반이형성되는병으로, 인삼지상부에서발생하는병중가장발생빈도가높으며발병시가장많은수량감소피해를초래한다 (Bae et al., 2005). 인삼점무늬병은주로 4월부터 8월까지발생하며, 발생정도는강우시기, 기간및강우량등과밀접한관련이있는것으로알려져있다 (Oh et al., 1987; Kang et al., 2007). 인삼점무늬병중줄기점무늬병은 4월하순부터발생하기시작하여 5월중순이후부터는감소하며, 잎점무늬병은 5월중순부터발생이증가하여저년생에서는 6월중순에가장많이발생한다. 또한, 저년생보다는고년생에서심하게발생하며수분에잘노출되는전주에서더많이발생한다 (Cho et al., 1998). 점무늬병, 잿빛곰팡이병등인삼에서발생되는병해방제를위해서물리적방제, 경종적방제및화학적방제등여러방제방법을이용하고있으나, 대부분사용이편하고효과가빠른화학적방제방법을주로사용하고있다 (Lee et al., 2008). 최근화학농약의오남용으로인한토양, 수질등환경오염부작용및농산물의안전성에대한우려로잔류농약에대한규제가점차엄격해지고있는추세이다. 이에따라, 인삼재배농가에서농약잔류에대한우려없이사용할수있도록화학농약을대체할수있는친환경농자재개발에대한요구가점점커지고있다 (Lee, 2012). 이와같이, 친환경농자재에대한관심과수요가증가함에따라인삼뿌리썩음병, 모잘록병등뿌리부위에서발생되는지하부병과점무늬병, 탄저병등잎, 줄기부위에서발생되는지상부병의친환경방제를위해 Cylindrocarpon destructans, Fusarium solani, Rhizoctonia solani, Pythium spp, Alternaria panax 등발병원인균에대해길항활성이있는미생물을활용하기위한연구가활발히진행되고있다 (Lee et al., 2008; Kim et al., 2012; Lee et al., 2012; Kim and Park, 2013; Lim et al., 2014; Lee et al., 2017). 본연구는인삼재배시인삼지상부병중발생빈도가높으며가장큰생산량감소를일으키는점무늬병방제를위해인삼재배지토양으로부터선발한길항미생물의화학농약대체가능성을분석하고친환경농자재로의개발가능성을확인하고자수행하였다. Materials and Methods 균주분리인삼점무늬병원인균 (Alternaria panax) 에길항활성을가진미생물분리를위해, 강원도철원군근남면인삼재배지표토층아래 10 cm 깊이의토양을채취하였다. 채취한토양 1 g을 9 ml의멸균수에넣고 25 C, 100 rpm 조건으로 30분동안진탕후십진희석한다음미리준비해놓은 TSA (Tryptic soy agar, Difco, USA) 배지에 200 µl씩도말하여 30 C에서 24-48시간동안배양하였다. TSA배지에서자라난단일콜로니를분리하여사면배지에서보존하면서인삼병원균에대한길항활성을검정하였다. 항균활성검정 토양에서분리한 KL87 균주의인삼점무늬병병원균에대한길항활성을확인하기위하여, PDA

3 Suppressive Effect of Bacillus amyloliquefaciens KL87 on Alternaria Blight of Ginseng 493 (Potato dextrose agar, Difco, USA) 배지에서 25 C, 10일간배양한 A. panax 균의균총가장자리를 cork borer로떼어낸후 PDA 배지가들어있는페트리디쉬에접종하였다. A. panax 균을접종한지점에서 2.5 cm 떨어진 PDA 배지위에 LB broth (tryptone 10 g, yeast extract 5 g, sodium chloride 10 g L -1 ) 에서 2일동안배양한분리균주콜로니를멸균수에희석하여 10 5 CFU/ml 농도로조절한다음, 희석한현탁액 10 µl를 paper disk에접종하였다. 항균활성검정을위한 A. panax 균주는농업유전자원센터에서분양받아보존하면서사용하였으며, 토양에서분리한균주의길항력은 25 C에서 10일간배양한후나타난저지원 (Inhibition zone) 의크기로확인하였다. 균주동정인삼점무늬병원균에대한길항활성을나타내는 KL87 균주의동정을위해유전적특성과생화학적특성을분석하였다. 유전적특성은 16S rrna 유전자분석을수행하였으며, 균주를 LB 배지에서배양후 8,000 rpm 으로 10분간원심분리하여균체를수확한다음, 균체 50 mg을 Genomic DNA Prep Kit (Solgent, Daejeon, Korea) 를이용하여 genomic DNA를추출하였다. 16S rrna 유전자증폭은 universal primer인 27F (5'-AGAGTTTGATCAT- GGCTCAG-3') 와 1492R (5'-GGATACCTTGTTA-CGACTT-3') 을이용하였다. PCR 반응은정방향프라이머 (27F) 1 µl, 역방향프라이머 (1492R) 1 µl, DNA 20 ng/µl, 4dNTP (10 mm) 2µl, 10 Taq 버퍼 2.5 µl, solgent EF Tag 0.3 µl, 멸균수 17.2 µl가혼합된총 25 µl의 PCR 혼합반응액을 94 C, 20초동안가열하여 DNA의변성을유도하였다. 변성유도후에는반응온도를 50 C로낮추어프라이머가일치하는 sequence에결합할수있도록 40초동안반응시켰으며, 이후 72 C에서온도를높여 DNA 복제를 1분 30초동안유도하는주기 (cycle) 를 1cycle로하여 30 cycle을실시하였다. 염기서열분석은 Solgent Co. (Daejeon, Korea) 에의뢰하여분석하였으며, 바실러스속표준균주의염기서열은미국국립생물정보센터 (National Center for Biotechnology Information) 에서받아 MEGA 4.0 program으로계통도를작성하였다 (Tamura et al., 2011). 계통도는 neighbor joining 알고리즘을이용하여작성하였고 Bootstrapping을 1,000회반복하여견고성을확인하였다. 길항균주의생화학적특성은 Vitek2 compact system (biomérieux, France) 을이용하여 46가지의특성을분석하였다. 순수배양된균주를 0.45% NaCl이함유되어있는생리식염수에혼합하여 Vitek densichek (biomérieux, France) 로탁도 2 McFarland로현탁한후, BCL card (biomérieux, France) 에분주하여 Vitek2 system에서생화학특성을분석하였으며, 생화학특성분석후 Vitek2 Advanced Expert System (AES) software로판독하여균주를동정하였다 (Lim et al., 2015). 인삼점무늬병방제효과검정선발한 KL87 균주의인삼재배지에서의점무늬병발생억제효과검정을위해시험시료를다음과같이제조하였다. 시험균주를 LB broth (Difco, USA) 에접종한다음진탕배양기 (SK-760M, Jeiotech, Korea) 에서 25 C, 120 rpm 조건으로 3일동안배양하여얻은배양액에 skim milk (Difco, USA) 10% 를넣고 5일동안동결건조하였다 (FD8508, Ilshin, Korea). 동결건조한 KL87균주의생균수는동결건조한시료 1 g을단계별로희석한후배양한다음균수를조사하여측정하였으며, 점무늬병발생억제효과는동결건조한시험균주를 CFU/mL농도로희석하여사용하였다. KL87 균주의인삼점무늬병방제효과검정시험은 4년근인삼재배지에서수행하였으며 5월하순에서 7월상순사이재배면적 1.62 m 2 당균주희석액 1 L씩을 10-15일간격으로 4회엽면살포하였다. 방제효과비교를위한대조구는 Polyoxin B 1,000배희석액, Mancozeb 600배희석액및 Polyoxin D 1,000배희석액을 5월하순에서 7월상순사이재배면적 1.62 m 2 당 1 L씩 4회교호살포하였으며, 방제효과는점무늬병이병엽을조사하여방제가 (%) 를아래의공식에따라계산하여최종분석하였다.

4 494 Korean Journal of Soil Science and Fertilizer Vol. 51, No. 4, 2018 방제가 (%) = {( 무처리군발병도 - 방제처리군발병도 ) 무처리군발병도 } 100 Results and Discussion 균주분리및항균활성검정인삼점무늬병방제를위한길항균선발을위해인삼재배지토양시료에서 260여균주를분리하여인삼점무늬병원인균 (A. panax) 에대한각균주의항균활성을조사하였다. 분리한균주중 34개의균주가점무늬병원균의생장을억제하는효과를가지고있었으며 (data not shown), 이중항균활성이우수한 KL87 균주를최종선발하여유전적특성및생화학적특성을분석하였다. KL87균주는인삼점무늬병원균인 A. panax균의생장을강하게억제할뿐만아니라인삼잿빛곰팡이병원균 Botrytis cinerea의생장도억제하는활성을가지고있었다 (Fig. 1). 잿빛곰팡이병은인삼재배시잎과지제부줄기등에발생하여큰피해를입히는병으로, 7월중순부터발병하여 8월중순이후에는발병율이높아지는주요병해로서 (RDA, 2009), KL87 균주는인삼재배시발생하는점무늬병및잿빛곰팡이병등주요지상부병을동시에효과적으로방제할수있을것으로판단되며, KL87 균주를이용한친환경방제제개발가능성을확인할수있었다. Fig. 1. Antifungal activity of Bacillus amyloliquefaciens KL87 strain on growth of Alternaria panax (A) and Botrytis cinerea (B). AP: Alternaria panax, BC: Botrytis cinerea. 분리균주동정인삼점무늬병원균에길항활성이있는 KL87 균주의정확한동정을위해 16S ribosomal RNA 유전자염기서열을분석한후분자계통도를작성하였다. KL87 균주는 Bacillus amyloliquefaciens subsp. plantarum strain FZB42 및 Bacillus amyloliquefaciens strain NBRC15535와 99% 의높은상동성을보였으며, 염기서열을바탕으로한계통수분석결과, Bootstrap 값이 72% 로비교적높은것으로확인되었다 (Fig. 2). 독성을가진항균성물질을분비하여길항활성을나타내는것으로알려져있는바실러스 (Bacillus) 속미생물을이용하여인삼재배시수량감소피해를야기하는지상부병을방제하기위한연구가활발히진행되고있으며, Bacillus subtilis를이용한인삼점무늬병방제 (Lee et al., 2008), 인삼점무늬병원균 (Alternaria panax) 과탄저병원균 (Colletotrichum gloeosporioides) 에대한길항활성을가진 Bacillus균 (Lee et al., 2012) 등이보고된바있다. KL87 균주의 16S ribosomal RNA 염기서열을분석한결과, B. amyloliquefaciens와높은상동성을보여 B. amyloliquefaciens속하는균주인것으로추정되었으나, 정확한균주확인을위하여생화학적특성을분석하였다. Vitek 2 system을이용한생화학적특성검정결과 (Table 1), KL87 균주는 β-xylosidase, Phenylalanine arylamidase 및 L-Pyrrolydonyl-arylamidase 등 18종류의생

5 Suppressive Effect of Bacillus amyloliquefaciens KL87 on Alternaria Blight of Ginseng 495 Fig. 2. Phylogenic tree based on 16S rrna gene sequences showing relationships between KL87 strain and the species of the genus Bacillus. The numbers at the nodes are the percentage bootstrap based on 1,000 resampled data set. Bar, 0.1 substitutions per nucleotide position. Table 1. Biochemical characteristics of KL87 strain. Biochemical test Results Biochemical test Results β-xylosidase + D-Mannitol + L-Lysine-arylamidase - D-Mannose + L-Aspartate arylamidase - D-Melezitose - Leucine arylamidase - N-Acetyl-D-Glucosamine - Phenylalanine arylamidase + Palatinose + L-Proline arylamidase - L-Rhamnose - β-galactisidase - β-glucosidase + L-Pyrrolydonyl-arylamidase + β-mannosidase - α-galactisidase + Phosphorylcholine - Alanine arylamidase + Pyruvate + Tyrosine arylamidase - α-glucosidase - β-n-acetyl-glucosaminidase - D-tagatose - Ala-Phe-Pro arylamidase + D-trehalose + Cyclodextrine - Inulin - D-Galactose - D-glucose - Glycogene - D-ribose - myo-inositol - Putrescineassimilation - Methyl-A-D-Glucopyranoside acidification + Growth in 6.5% NaCl + Ellman - Kanamycin resistance - Methyl-D-xyloside - Oleandomycin resistance - α-mannosidase - Esculinhydrolyse + Maltotriose - Tetrazolium red + Glycine arylamidase + Ploximin B resistance +

6 496 Korean Journal of Soil Science and Fertilizer Vol. 51, No. 4, 2018 화학반응에서양성을나타냈으나 L-Lysine-arylamidase, L-Aspartate armylamidas 및 Leucine arylamidase 등 28 종류의생화학반응은음성을나타내어 Bacillus amyloliquefaciens와 99% 유사한생화학적특성을가진것으로확인되었다 (Table 2). Bacillus amyloliquefaciens 균은식물체의뿌리주위나식물내에서흔하게발견되는미생물로서고추역병균인 Phytophthora capsici의생육을억제하며 (Korea patent, ), 인삼모잘록병원균인 Phytium ultimum과 Rhizoctonia solani의생장억제활성을가지고있는것으로보고된바있고 (Park et al., 2016), 인삼뿌리썩음병원균 (Cylindroncarpon destructans) 및모잘록병원균 (Rhizoctonia solani) 등에도항균활성이있는것으로알려져있다 (Kim et al., 2012; Jamal et al., 2015). 이와같이, A. panax와 B. cinerea 생장억제활성을가지고있는 KL87균주는유전적, 생화학적특성분석을통해 Bacillus amyloliquefaciens 균으로확인되었으며, Bacillus amyloliquefaciens 균은여러연구자들에의해식물병원균에대한항균효과가우수한것으로보고되고있어친환경농자재로개발시인삼에서발생하는점무늬병등의지상부병을효과적으로방제할수있을것으로판단된다. Table 2. Identification of the isolated KL87 strain by Vitek2 system. Identification Result Strain Note Species Probability KL87 Bacillus amyloliquefaciens 99% Identified 인삼점무늬병방제효과검정동결건조한 KL87 균주를 CFU/mL농도로희석한후 4년근인삼재배지에 5월부터 7월까지 10-15일간격으로총 4회엽면살포한후점무늬병발생억제효과를분석하였다. 점무늬병방제효과시험결과, 무처리구의점무늬병발병률은 24.8±3.8% 였으나, Polyoxin B 1,000배액등화학농약을교호살포한시험구는발병률이 2.2±1.1% 로 91.3% 의방제효과를나타냈으며, KL87 균주를엽면살포한시험구의점무늬병발병률은 6.0±0.9% 로무처리구대비 75.9% 의방제효과를나타냈다 (Fig. 3). KL87 균주의인삼점무늬병방제효과는화학농약대비 83% 수준으로효과가매우우수하여친환경농자재로의개발및산업화가능성을확인할수있었다. KL87 균주의처리적기확인을위해 5월과 6월로미생물제처리시기에차이를두어점무늬병방제효과를시험하였다. 시험결과, 5월부터화학농약처리시점무늬병발병률은 2.2% 로무처리구의 24.8% 대비 91.3% 의방제효과를나타냈으나, 6월처리시 77.2% 의방제효과를나타내어 5월처리구에비해방제효과가 14.1% 감소하였다. KL87 균주는 6월부터처리시점무늬병방제율이 38.2% 로 5월처리구방제율 75.9% 에비해큰폭으로감소하였으며, 이와같은결과로보아 KL87 균주를이용한인삼점무늬병의효과적인방제를위해서는발생초기인 5월부터미생물제를처리하는것이적절한것으로판단된다 (Fig. 4). 최근, 안전농산물선호및잔류농약규제강화추세에따라농약잔류에대한우려없이사용할수있는친환경농자재개발에대한연구가활발히진행되고있다. 인삼점무늬병방제효과가우수한 KL87 균주는향후균주최적배양을위한생리특성및방제효과를최대화할수있는제형화기술등에대한추가적인연구를통해농업현장에서화학농약을대체할수있는친환경농자재로활용가능할것으로사료된다.

NT: no treatment, Polyoxin B: alternate application (Polyoxin B, Mancozeb and Polyoxin D), KL87: treated with Bacillus amyloliquefaciens KL87 at a concentration of

7 Suppressive Effect of Bacillus amyloliquefaciens KL87 on Alternaria Blight of Ginseng 497 Fig. 3. Suppressive effect of Bacillus amyloliquefaciens KL87 on Alternaria blight occurrence in the field of ginseng cultivation. NT: no treatment, Polyoxin B: alternate application (Polyoxin B, Mancozeb and Polyoxin D), KL87: treated with Bacillus amyloliquefaciens KL87 at a concentration of CFU/mL. Fig. 4. Control value of Bacillus amyloliquefaciens KL87 on Alternaria blight of ginseng according to beginning time of treatment. Polyoxin B: alternate application (Polyoxin B, mancozeb and Polyoxin D), KL87: treated with Bacillus amyloliquefaciens KL87.

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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). 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