13(이동운)p fm

Weed Turf. Sci. 2(2):202~206, 2013 DOI:dx.doi.org/10.5660/WTS.2013.2.2.202 Print ISSN 22877924, Online ISSN 22883312 Research Note Open Access Weed & Turfgrass Science The Korean Society of Weed Science and The Turfgrass Society of Korea Rhizoctonia solani AG22 IV 에대한 Helicosporium sp. KCTC 0635BP 의항균활성 이상명 1 김동수 2 이광수 2 이총규 3 이동운 1 * 1 경북대학교생태환경관광학부생물응용전공, 2 국립산림과학원남부산림자원연구소, 3 경남과학기술대학교산림자원학과 Antibiotic Properties of Helicosporium sp. KCTC 0635BP to Rhizoctonia solani AG22 IV Sang Myeong Lee 1, Dong Soo Kim 2, KwangSoo Lee 2, ChongKyu Lee 3, and Dong Woon Lee 1 * 1 Major of Applied Biology, School of Ecological Environment and Tourism, Kyungpook National University, Sangju, Gyeongbuk, 742711, Republic of Korea 2 Southern Forest Resources Research Center, Korea Forest Research Institute, Jinju, 600300, Republic of Korea 3 Department of Forest Resources, Gyeongnam National University of Science and Technology, Jinju, Gyeongnam, 660 758, Republic of Korea (Received on June 10, 2013; Revised on June 17, 2013; Accepted on June 18, 2013) ABSTRACT. Biocontrol potential of an isolate of Helicosporium spp. against Rhizoctonia solani, Fusarium oxysporium and Phytophthora drechsleri was evaluated in vitro and in vivo. A selected biocontrol agent designated as Helicosporium 0635BP strongly inhibited growth and lysed mycelium of Rhizoctonia solani and Fusarium oxysporium on PDA. Autoclaved culture filtrate of the agent also completely inhibited growth of the turfgrass large patch pathogen, R. solani AG22 IV at the concentration of 50 ml L 1. The pathogen was killed when dipped under the 20% filtrate for four hours or 50% for one hr. In a field trial, plots applied with the crude or times diluted culture filtrate showed 100% control efficacy of the turfgrass large patch as a chemical applied for a comparison. Results indicated that Helicosporium 0635BP is a promising biocontrol agent on control of the turfgrass large patch disease and its culture filtrate contained unknown heat suitable antifungal substance (s). Further studies on mass production, purification and identification of the unknown compound (s) are in progress for practical use. Key words: Biocontrol, Helicosporium, Large patch, Turfgrass 토양등의자연생태계로부터유용미생물을분리하고선발하여이들을식물병해충방제에이용하는생물적방제는국내외적으로많은관심을받는연구분야이다 (Elad et al., 1994). 각종식물병에대한생물적방제연구는병원균에대해길항작용을나타내는유용미생물을식물의근권에정착시켜토양전염성병원균을사멸시키거나밀도증가를억제시킴으로써병원균의식물체침입을제어하는직접적인방법과이들이분비하는항균성물질을식물병방제수단으로이용하는간접적인방법등이있다 (Mandeel *Corresponding author: Phone) +82545301212, Fax) +82545301218 Email) whitegrub@knu.ac.kr and Baker, 1993; Omura et al., 1988). 각종작물의식물병억제를위하여다양한종류의길항미생물들이선발되어활용되고있는데이러한길항균들의길항작용은항생이나경합, 기생, 균사간섭, 용균등의형태로나타나고있다 (Back et al., 2001). Rhizoctonia solani AG22 IV에의해들잔디류 (Zoysia spp.) 에발생하는잔디갈색퍼짐병 (large patch) 은우리나라에서늦봄과가을 2회에발생하여원형의마름증상을나타내는데발병초기에는잔디잎이황갈색으로변하나이후원형의마름현상을나타낸다. 병반의중심부잔디잎은회갈색으로변하며병반가장자리진전부위는황갈색으로변하는데발병면적이넓고, 병원균이토양댓취층에분포하고, 장마기나배수불량지에서발병이잘되어방제가 202

Rhizoctonia solani AG22 IV 에대한 Helicosporium sp. KCTC 0635BP 의항균활성 203 어려운식물병의하나이다 (Shim et al., 1994; Jung et al., 2008). 갈색퍼짐병이골프장에서잔디의품질을심각하게위협하는병이기때문에관리자들은집약적인관리를하고있으며주로화학농약에의존하고있다. 그러나이러한집약적인화학적방제는약제저항성의발현과환경과인축에대한부작용등이우려되고있으며공원지나경기장잔디에농약을처리하는것에대한일반인들의거부감도높은실정이다 (Shim and Kim, 2000; Uddin and Viji, 2002). 때문에골프장잔디에서갈색퍼짐병을방제할수있는화학적방제대체법강구를위하여다양한연구들이수행되고있는데목초액과같은천연물제제나길항미생물을이용한생물적방제, 저항성품종의개발등이시도되고있다 (Chai et al., 2001; Geon et al., 2005; Islam et al., 2009; Jung et al., 2008). Helicosporium은목재부후균으로소나무류 (Pinus), 포플라 (Populus), 유칼립투스 (Eucalyptus) 등의고사목에서부생적으로기생하는곰팡이로알려져있다 (Goos, 1987). Helicosporium이목재를부식시켜영양원을흡수 이용하기 위해서목재의주구성분인 cellulose와 pectin을분해하는 cellulase나 pectinase를분비할것으로알려져있다 (Goos, 1987). 일반곰팡이의세포벽주성분은식물과같이 cellulose 로구성되어있으므로 Helicosporium은다른곰팡이의세포벽을분해시키거나형성을억제시키는길항작용을나타낼것으로생각되는데 Hardy and Sivajithanparam (1995) 는 Helicosporium으로부터항생능력을처음으로발견하였으며 Choi et al. (2012) 는본연구에이용된균주로부터항생물질 2methylresorcinol을확인한바있다. 그러나, Helicosporium 을직접토양에처리하여토양전염성병을방제하거나대사산물을이용한식물병방제연구는아직보고된바없는실정이다. 따라서본연구자들은밤나무고사목으로부터분리한 Helicosporium의주요토양전염성식물병원균인 Rhizoctonia solani와 Fusarium oxysporium 및 Phytophthora drechsleri 등에대한항균활성여부를실내에서검정하고, 배양상등액이병원균에미치는영향을조사하였으며, 포장에서잔 디갈색퍼짐병방제효과를조사하여생물적방제이용가능성을검토하였다. 식물병원균의균사생장억제효과본실험에사용된 Helicosporium sp. KCTC 0635 균주는 1998년경남하동의밤나무고사목에서분리되었으며, 균사는나선모양을나타내고, 배양적특징으로는 PDB배지에서 1주간배양시밤색의색소를분비한다. 따라서본시험균주는 Helicosporium sp. 으로동정되었다 (Choi et al., 2012; Goos, 1987). 분리균의항균활성은감자한천배지 (PDA) 에서 Helicosporium sp. KCTC 0635를배지가분주된 plate 한편에접종하여 25 o C에서 5일간배양한후오이모잘록병원균 (Rhizoctonia solani), 잔디갈색퍼짐병원균 (Rhizoctonia solani AG22 IV), 시들음병원균 (Fusarium oxysporium) 및역병균 (Phytopthora dreschsleri) 의균총을 40 mm 거리에대치배양하였다. 각병원균의최적배양온도에서 7일간배양후두균사이의저지원크기를조사하였다. 한개의 plate를한반복으로 3반복실험하였다. Helicosporium sp. KCTC 0635 BP 균주는오이에서분리된 Rhizoctonia solani와잔디에서분리된 Rhizoctonia solani AG22 IV 균에대해서매우높은길항력을나타내었다 (Table 1). R. solani (cucumber) 에대한저지원은 15 mm, R. solani AG22 IV는 12 mm 였다. 그러나오이의주요토양전염병균인 Fusarium oxysporium과 Phytophthora dreschsler 에대한항균활성은상대적으로낮거나없는것으로나타났다 (Table 1). 공시한모든병원균에대한 coiling 현상은보이지않았으며, 균사의 lysis는 Phytophthora dreschsler 을제외한 3종의병원균에대하여보였다. 배양여액의농도별식물병원균에대한억제효과. 분리균의배양액을얻기위하여 850 ml 병버섯재배용플라스틱용기에 PDB 500 ml를넣어 121 o C 1.5기압에서 30분간멸균하였다. PDA배지에서 7일간배양한분리균의균사절편 ( 직경 :10 mm) 을미리멸균하여준비한 PDB가분주된버섯재배용기에접종후 25 o C 진탕배양기 (120 rpm) Table 1. Antagonistic activity of Helicosporium sp. KCTC 0635BP to Rhizoctonia solani, Rhizoctonia solani AG22 IV, Fusarium oxysporium and Phytopthora dreschsleri. Fungi Host Antagonism Coiling Lysis Over growing Inhibition zone (mm) a Rhizoctonia solani Cucumber + 15 Rhizoctonia solani AG22 IV Turf grass + 12 Fusarium oxysporium Cucumber + 5 Phytophthora dreschsler Torreya a Inhibition was distance between pathogen and Helicosporium sp. KCTC 0635BP.

204 이상명 김동수 이광수 이총규 이동운 Table 2. Effect of culture filtrate of Helicosporium sp. KCTC 0635BP on the mycelium growth of Rhizoctonia solani (cucumber), R. solani AG22 IV and Fusarium oxysporium. Concentration (%) R.solani (Cucumber) R. solani AG22 IV F. oxysporium Diameter (mm) % Control x Diameter (mm) % Control Diameter (mm) % Control Control 1 5 10 20 50 100 63.3 48.7 17.3 11.3 6.3 23.1 c 72.7 b 82.2 ab 90.1 a 28.3 26.3 7.1 b 15.0 16.0 16.0 14.0 14.0 11.0 5.0 d d 6.7 c 6.7 c 26.7 b 66.7 a x The same letter in column represents not significantly different (P=5 : Duncan's multiple range test). % Control={(Mycelial growth(mm) on controlmycelial growth(mm) on treatment)/mycelial growth(mm) on control} 100. 에서 10일간배양하여 121 o C 1.5기압에서 30분간멸균하였다. 멸균후킴와이프스로균사를제거하여배양여액 ( 조배양여액 ) 을얻었다. PDA배지에배양여액을 1, 5, 10, 20, 50 및 100% 비율로첨가하여살균한다음 9 cm Petri dish 에 20 ml씩분주하였다. PDA배지에서 5일간배양한 Rhizoctonia solani (cucumber), Rhizoctonia solani AG22 IV 및 Fusarium oxysporium 균사절편 ( 직경 : 5mm) 을접종하여배양 7일후균총의직경을측정하였다. 처리는한개의 Petri dish를한반복으로 3반복으로하였다. Helicosporium sp. KCTC 0635 BP와식물병원균과의대치배양시길항작용이매우높은것으로확인되어 Helicosporium sp. KCTC 0635 BP의배양여액을농도별로첨가한배지에서의억제효과를알아본결과는 Table 2와같았다. Helicosporium 의배양여액은식물병원균의종류와배양여액의농도에따라억제율에큰차이를보였다. 잔디갈색퍼짐병균에대한억제율이가장높게나타나배양여액 5% 처리농도에서도 100% 의억제율을보였다. 오이잘록병균에대한항균활성은 5% 농도에서 70% 이상의억제율을보였고, 50% 농도에서는 100% 의억제율을보였다. 시들음병균에대해서는 100% 농도에서 66.7% 의억제율을보여 Rhizoctonia solani에비해억제효과가떨어지는경향을보였다. Rhizoctonia solani AG22 IV에대한항균효과. Helicosporium sp. KCTC 0635BP 균배양여액의 Rhizoctonia solani AG22 IV에대한농도별및침지시간에따른항균효과를알아보기위하여배양여액을 1, 5, 10, 20, 50, 100% 비율로조절하여 50 ml 삼각플라스커에 20 ml를넣어살균하였다. PDA배지에서배양한 Rhizoctonia solani AG22 균사절편 ( 직경 : 5 mm) 을 1, 4, 8, 24시간침지시킨후살균수로배양여액을제거하여 PDA배지 ( 직경 9 cm Petri dish) 에접종하여 25 o C항온기에서배양하여균총의직경을측정하였다. 처리는한개의 Petri dish를한반복으로 3반복 으로하였다. Helicosporium sp. KCTC 0635 BP 균주의배양여액농도와침지시간에따른잔디갈색퍼짐병균에대한항균효과는 Table 3과같았다. 침지시간이길수록낮은농도에서도높은억제율을보여 100% 농도에서는 1시간, 50% 농도에서는 3시간, 20% 농도에서는 24시간침지시 100% 의억제율을보였다. 갈색퍼짐병방제효과배양여액을이용한잔디라지패취병의방제효과를알아보기위하여 5월부터 6월까지부산의 A골프장 11번홀들잔디 (Zoysia japonica) 에 PDB에서배양한배양여액을 1배, 10 배, 150배, 500배액으로살포하였다. 처리전잔디갈색퍼짐병병반의크기를측정한후, 병반가장자리에 4방향으로표시를하였고, 처리 7일후병반크기를측정하였다. 방제효과는처리전발생면적과처리후발생면적을조사하여병진전율을구하였다. 대조약제로는잔디의갈색퍼짐병방제약제로등록되어있는 Pencycuron 25 WP를기준량으로처리하였다. 실험은 3반복으로수행하였고, 약제처리전후강우는없었다. 통계처리는처리평균간차이를 Duncan s multiple range test (P<5) 로분산분석하였다. Helicosporium sp. 배양여액 100% 와 10배처리에서잔디갈색퍼짐병에대하여 100% 의방제가를나타내어대조약제와동일한효과를나타내었다 (Table 4). 또한처리후지속기간에있어서도배양여액을처리한경우에는 30일후까지도병진전이없었으나농약을처리한경우에서는 30일후병이진전되는것을관찰할수있었다. Helicosporium의항균활성에대한연구는매우제한적이며 1995년 Hardy와 Sivasithamparam에의해처음언급되었다. 본연구에사용한 Helicosporium 0635BP 균주의배양액에서는 2methylresorcinol의존재가확인되었으며이

Rhizoctonia solani AG22 IV 에대한 Helicosporium sp. KCTC 0635BP 의항균활성 205 Table 3. Antagonistic activity of Helicosporium sp. KCTC 0635BP according to dipping time of large patch on culture filtrate of Helicosporium sp. KCTC 0635BP. Concentration of culture filtrate (%) Dipping time (hr) Diameter(mm) of R. solani AG 22 IV % Control x 2 days 4 days 2 days 4 days 0 1 27.0 85.0 4 27.1 85.0 8 27.8 85.0 24 27.5 85.0 1 1 27.0 65.0 b y 23.5 b 4 26.0 62.0 4.1 ab 27.1 b 8 24.7 61.7 11.2 a 27.4 b 24 24.0 52.0 12.7 a 38.8 a 5 1 26.0 6 3.7 a 29.4 a 4 26.0 62.0 4.1 a 27.1 a 8 27.0 6 2.9 a 29.4 a 24 25.7 59.1 6.6 a 30.5 a 10 1 25.2 53.0 6.7 b 37.7 a 4 23.0 58.0 15.1 ab 31.8 a 8 24.4 59.2 22.2 a 30.4 a 24 24.7 58.0 10.2 b 31.8 a 20 1 16.0 43.0 40.7 b 49.4 b 4 8 24 50 1 4 8 24 100 1 4 8 24 x Control (%) = (1treatment/check) 100. y The same letter in the column is not significantly different (P=5 : Duncan's multiple range test). 물질은그람양성균들에대한저해활성이그람음성균들에비하여상대적으로높게나타났으며사람의폐암세포주 MCF7에대해서도최소저해농도가 5.4 µg/ml로낮게나타났다 (Choi et al., 2012). 그러나 Helicosporium 0635BP 균주의식물병에대한영향은보고된바없으며본균주에서항균력이있는물질로확인된 2methylresorcinol이 Table 4. Effect of culture filtrate of Helicosporium sp. KCTC 0635BP on development of large patch by Rhizoctonia solani AG22 IV at 7 days after treatment in field experiment. Treatment Area of patch (%) % control x Control Antagonistic filtrate 1 10 150 500 Pencycuron 25 WP 식물병원균에대한항균활성은검토된바없다. 본연구의결과 Helicosporium 0635BP 균주는오이의모잘록병균인 Rhizoctonia solani와잔디의갈색퍼짐병균 R. solani AG22 IV에대한균사성장억제가역병균이나시들음병균에비하여높게나타났으며같은 Rhizoctonia solani중잔디갈색퍼짐병에대한항균활성이더높게나타났다. 한편본균주는살균한배양여액이이들병원균에대한높은항균활성을보임으로인해활성물질이열에매우안정적인물질로추정되며활성물질이생균류에포함된것이아니어서실용적인측면에서유용할것으로판단된다. 특히골프장들잔디에서실험한결과 10배희석액에서까지 100% 의방제효과를보여항균활성성분의동정을통해유효성분의함량을높인다면생화학농약으로사용가능성도충분할것으로판단된다. 또한본균주의배양여액은야외실험에서잔디에대하여약해를나타내지않았으며오이를대상으로발아실험과생육실험을수행한결과도약해가없는것으로식물체에안전한것으로판명되었다 ( 자료미제시 ). 특히대조약제로사용한화학농약에비하여 30일이후까지잔디갈색퍼짐병에대한억제효과가있는것으로나타나잔디밭에서화학농약에비하여상대적으로지속성이높은것으로판단되었다. 또한부생성이강하여일반배지상에서다른길항미생균들에비하여쉽게증식이되는특성을가지고있어저가의배양원탐색이수반된다면배양비용절감으로대량생산을위한경제성확보가가능할것으로판단된다. 추후본균주의 Rhizoctonia solani에대한항균물질분석과활성력검정이필요할것으로생각된다. 요 23.5 18.6 25.2 약 y 20.9 b c x Control (%) = (1treatment/check) 100. y The same letter in column are not significantly different (P=5 : Duncan's multiple range test). Helicosporium spp. 의식물병원균 Rhizoctonia solani와 Fusarium oxysporium, Phytophthora drechsleri에대한항균

206 이상명 김동수 이광수 이총규 이동운 활성을실내와야외에서검정하였다. Helicosporium 0635BP 균주는 Rhizoctonia solani와 Fusarium oxysporium에대해 PDA배지상에서강한균사억제효과를보였다. 살균한배양여액 50 ml/l 처리시잔디갈색퍼짐병균 (Rhizoctonia solani AG22) 에대한높은생장억제효과를보였다. 배양여액 20% 에 4시간동안침지하거나 50% 배양여액에 1시간침지시 Rhizoctonia solani AG22 IV는 100% 사멸하였다. 골프장들잔디에서 Helicosporium 0635BP 균주의배약여액처리시 10배액까지잔디갈색퍼짐병의병진전을대조약제와동일하게 100% 억제하였다. 따라서 Helicosporium 0635BP 배양액은잔디갈색퍼짐병의생물적방제인자로활용가능할것으로생각되며실용화를위하여대량배양과항균물질의정제와동정이필요할것으로보인다. 주요어 : 갈색퍼짐병, 생물적방제, 잔디, Helicosporium. References Back, S.B., Gu, H.M., Do, E.S. and Cheon, S.C. 2001. Biocontrol of plant diseasemajor in natural plants and antagonistic microorganism. Jigu Publishing Co. Seoul, Korea. (In Korean) Chai, M.L., Lee, J.M., Park, M.H. and Kim, D.H. 2001. In vitro selection for large patch resistance in zoysiagrass. J. Kor. Soc. Hort. Sci. 42:249253. (In Korean) Choi, H.J., Lee, S.M., Kim, S.H., Kim, D.W., Choi, Y.W., et al. 2012. A novel Helicosporium isolate and its antimicrobial and cytotoxic pigment. J. Microbial. Biotechnol. 22:12141217. Elade, Y., Kohl, J. and Fokkema, N.J. 1994. Control of infection and sporulation of Botrytis cinerea on bean and tomato by saprophytic yeasts. Phytopathology 83:308313. Geon, M.G., Kim, I.S., Lee, S.C., Son, T.K., Shim, G.Y., et al. 2005. Effects of pyroligneous acid on control of large patch in zoysiagrass. Kor. Turfgrass Sci. 19:7383. (In Korean) Goos, R.D. 1987. Fungi with a twist: The helicosporous hyphomycetes. Mycologia 79:122. Hardy, E.S.J. and Sivasithamparam, K. 1995. Antagonism of fungi and actinomycetes isolated from composted Eucalyptus bark to Phytophthora drechsleri in a steamed and nonsteamed composted Eucalyptus barkamended container medium. Soil Biol. Biochem. 27:243246. Islam, M.R., Jeong, Y.T., Ryu, Y.J., Song, C.H. and Lee, Y.S. 2009. Isolation, identification and optimal culture conditions of Streptomyces albidoflavus C247 producing antifungal agents against Rhizoctonia solani AG22. Mycobiology 37:114120. Jung, W.C., Shin, T.S., Kim, B.S., Im, J.S., Lee, J.H., and Kim, J.W. 2008. Efficacy of antagonistic bacteria for bacteria for biological control of Rhizoctonia bright (large patch) on zoysiagrass. Res. Plant dis. 14:4350. (In Korean) Mandeel, Q. and Barker, R. 1993. Mechanisms involved in biological control of Fusarium wilt of cucumber with strains of nonpathogenic Fusarium oxyspotium. Phytopathology 81:615 621. Omura, S., Tomoda, H., Kimura K., Kumagai, H., Igarashi, K., et al. 1988. Atpenins new antifungual antibiotics produced by Penicillum sp. (production, isolation, physicochemical and biological properties). J. Antibiot. 41:17691773. Shim, G.Y. and Kim, H.G. 2000. Control of large patch caused by Rhizoctonia solani AG22 by combined application of antagonists and chemicals in golf courses. Kor. Turfgrass Sci. 13:131138. (In Korean) Shim, G.Y., Kim, J.W. and Kim, H.K. 1994. Occurrence of Rhizoctonia blight of zoysiagrasses in golf courses in Korea. Korean J. Plant Pathol. 10:5460. (In Korean) Uddin, W. and Viji, G. 2002. Biological control of turfgrass disease. pp. 313314. In: Gnanamanckam (ed.). Biological control of crop diseases. Marcel Dekker, Inc. Barsel, New York. USA.