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1 식물병연구 Research Article Open Access Res. Plant Dis. 21(2) : 50-57(2015) Meloidogyne incognita Streptomyces netropsis Characterization of Streptomyces netropsis Showing a Nematicidal Activity against Meloidogyne incognita 1, * 1, 2, 3, 4, 5 *Corresponding author Tel : Fax: kjinc@jnu.ac.kr Ja Yeong Jang 1,2, Yong Ho Choi 3, Yoon-Jung Joo 4, Hun Kim 3, Gyung Ja Choi 3, Kyoung Soo Jang 3, Chang-Jin Kim 4, Byeongjin Cha 2, Hae Woong Park 5 and Jin-Cheol Kim 1 * 1 Division of Food Technology, Biotechnology and Agrochemistry, Jeonnam National University, Gwangju , Korea 2 Department of Plant Medicine, Chungbuk National University, Cheongju , Korea 3 Eco-friendly New Materials Research Group, Korea Research Institute of Chemical Technology, Daejeon , Korea 4 Microbial Resource Center, Korea Research Institute Bioscience and Biotechnology, Daejeon , Korea 5 World Institute of Kimchi, an Annex of Korea Food Research Institute 675-1, Imamdong, Namgu, Gwangju , Korea Received April 30, 2015 Revised May 27, 2015 Accepted June 11, 2015 Control of nematode has become difficult owing to the restricted use of effective soil fumigant, methyl bromide, and other non-fumigant nematicides. Therefore, it is urgently necessary to develop microbial nematicide to replace chemical nematicides. In this study, the 50% aqueous methanol extraction solution of fermentation broths of 2,700 actinomycete strains were tested for their nematicidal activity against second stage of juveniles (J2s) of Meloidogyne incognita. As the results, only the 50% aqueous methanol extraction solution of AN110065, at 20% equivalent to 10% fermentation broth, showed strong nematicidal activity with 78.9% of mortality 24 h after treatment and 94.1% of mortality at 72 h. The 16S rrna gene sequencing showed that the strain sequence was 99.78% identical to Streptomyces netropsis. The extract of S. netropsis AN fermentation broth was successively partitioned with ethyl acetate and butanol and then the ethyl acetate, butanol and water layers were investigated for their nematicidal activity against the M. incognita. At 1,000 mg/ml, ethyl acetate layer showed the strongest activity of 83.5% of juvenile mortality 72 h after treatment. The pot experiment using the fermentation broth of AN on tomato plant against M. incognita displayed that it evidently suppressed gall formation at a 10-fold diluent treatment. The tomato plants treated with the fermentation broth of S. netropsis AN did not show any phytotoxicity. The results suggest that S. netropsis AN has a potential to serve as microbial nematicide in organic agriculture. Keywords : Biocontrol, Bio-nematicide, Meloidogyne incognita, Nematicidal activity, Streptomyces netropsis Research in Plant Disease The Korean Society of Plant Pathology pissn , eissn This is an open access article distributed under the terms of the Creative Commons Attribution License ( which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

2 Research in Plant Disease Vol. 21 No (Meloidogyne spp.). 11%, (McCart, 2009). 54% (Cho, 2000). 3 (M. arenaria), (M. hapla), (M. incognita), M. incognita M. arenaria (Kim, 2001). (Solanum lycopersicum L.),, M. incognita (Hwang, 2014; Qiao, 2011; Siddiqui Futai, 2009)... methyl bromide 2015 (Caboni, 2013; Oka, 2000), cadusafos (Qiao, 2011).. (Khan, 2008; Le Dang, 2011; Park, 2014; Siddiqui Mahmood, 1999; Tian, 2007) % (Berdy, 2005). Streptomyces 2. Streptomyces avermectin, milbemycin, bafilomycin, leucanicidins, geldanamycin, fungichromin B, fervenulin, 3-methoxy-2-methyl-carbazole-1,4-quinone, carbazomycins D F, 3-benzyl-1,4-diaza-2, 5-dioxobicyclo[4.3.0] nonane (Burg, 1979; Lacey, 1995; Nonaka, 2000; Ruanpanun, 2010; Ruanpanun, 2011; Skantar, 2005; Yoon, 2012; Zeng, 2013)., avermectin abamectin. Abamectin.. (M. incognita). in vitro in vivo. 방선균. Bennett (1% glucose, 0.1% yeast extract, 0.2% Bactopeptone, 0.1% beef extract, 2% agar) (Hesseltine, 1954) ml GSS (1% soluble starch, 0.025% K 2 HPO 4, 2.5% soybean meal, 0.1% beef extract, 0.4% yeast extract, 0.2% NaCl 2 g/l, 2% glucose, 0.2% CaCO 3 (ph7.2))(kim, 1989) 500 ml baffled 150 rpm, 28 o C 7. 분리균주의형태학적특성분석및균주동정. AN Bennett 28 o C 14. (Scanning electron microphotograph, SEM, JSM-6700F, JEOL Ltd, Japan). AN glutaraldehyde(2.5%, 0.1 M phosphate buffer, ph 7.0),. isoamyl acetate, CO 2 isoamyl acetate,. 16S rrna. AN ml Bennetts 28 o C, 150 rpm 3. 4 o C, 13,000 rpm 10. Qiagen DNeasy Blood & Tissue kit(qiagen, Hilden, Germany) DNA. 16S rrna universal primer(27f; 5 -AGAGTTTGATC- MTGGCTCAG-3, 1492R; 5 -GGTTACCTTGTTACGACTT-3 ) (polymerase chain reaction, PCR) (Lane, 1991). 16S rdna sequences

3 52 Research in Plant Disease Vol. 21 No. 2 16S rrna gene sequences CLUSTAL X software(thompson, 1997) MEGA (Tamura, 2013) Neighbour-joining method (Saitou Nei, 1987) Bootstrap 뿌리혹선충의증식및유충분리. Hwang (2014) ( ) (255 o C). (, ) 9.5 cm, 3. 10,000, ,. Barker (1985), 1 cm 0.5% (sodium hypochlorite) mesh 550 mesh. 550 mesh 3, Baermann funnel (Southey, 1986). 방선균의살선충활성스크리닝. 2, well tissue culture plate(becton Dickinson, Franklin Lakes, NJ). 1 ml o C, 13,000 rpm 10. (J2 ) 96-well tissue culture plate well 5, 10, 20% well 50 ml ( 50 J2). :(1:1, v/v) 20%. 96-well micro plate 30, 100% o C. 3, (Olympus CKX41, Tokyo, Japan), Abbotts formula (Abbott, 1925). (%) = [( - ) / (100 - )] 100. AN 균주용매분획의살선충활성. AN GSS 28 o C, 150 rpm ml, Whatman No o C (EYELA, Japan),.,,,,, 20 stock solution. 96-well tissue culture plate (M. incognita) 2 ( 50 /47.5 ml) 5% 100, 1,000 mg/ml. 5%. 3, 토마토포트에서뿌리혹선충병방제활성검정. AN (253 o C). 9.5 cm 400 g 2. 10,000. GSS 28 o C, 150 rpm 10 AN , 100, 1, (a. i. Fosthiazate 5%) 2,000 1,. 5, 6 Taylor Sasser(1978) (0, 0%; 1, 1-20%; 2, 21-40%, 3, 41-60%, 4, 61-80%, 5, %). 통계분석. SAS(SAS Institute, Inc, 1989, Cary, NC) (one-way analysis of variance)

4 Research in Plant Disease Vol. 21 No. 2 ANOVA 분석하였으며, Duncan s multiple range test(p = 0.05) 를 이용하여 유의성 검정을 수행하였다. 결 과 스크리닝을 통해 선발된 방선균의 살선충 활성. 2,700개 Fig. 1. Nematicidal activity of the methanol extract of AN against Meloidogyne incognita. Each value represents the mean ± standard deviation of three replicates. Bars indicate significant differences at 5% probability level. 53 의 방선균 배양액 메탄올 추출물중에 단 한 개 균주(AN11006) 추출물만이 고구마뿌리혹선충에 대해 높은 살선충 활성을 보 였다. AN 균주는 배양액 추출액 10% 처리 시 24시간 후 68.3%, 20% 처리 시 78.9%의 살선충율을 보였으며, 5% 처리구 에서는 거의 활성을 나타내지 않았다(Fig. 1). 또한, 시료 처리 72시간 후 10% 처리 시 73.2%의 활성을 나타냈으며, 20% 처리 시 94.2%의 살선충 활성을 보여 처리시간이 경과함에 따라 살 선충 활성이 유의성 있게 증가하였다(Fig. 1). 하지만 AN 균주의 배양여액만을 처리했을 때는 활성을 보이지 않았다. 무 처리 대조구로 멸균수:메탄올(1:1, v/v) 용액만을 20% 처리 했을 때 치사활성은 없었다. AN 균주의 형태학적 특성 및 16S rrna sequencing 분석. AN 균주는 흰색의 포자를 형성하였으며, 주사 전자현미경으로 관찰한 결과 rectiflexible 한 포자체인 형태를 보이고 원통형 모양의 mm 크기의 포자를 형성하였다 (Fig. 2A와 B). 16S rrna 유전자 염기서열에 기초한 분자 계통학적 분석 결 과, AN 균주는 Streptomyces netropsis NBRC 3723과 Fig. 2. Culture plate image (A) and scanning electron micrograph (B) of spore surface and hyphae (Bar = 10 mm) of AN on Bennett s agar at 28oC for 14 days. Neighbour-joining phylogenetic tree based on 16S rrna gene sequences showing the relationship between strain AN and 10 species of the genus Streptomyces. Numbers at nodes indicate levels of bootstrap support (%) based on analysis of 1000 resampled datasets; only values above 50% are given. NCBI accession numbers for each sequence are in parentheses. Bar, 2 substitution per 1000 nt 김진철o.indd 오후 2:38:56

54 Research in Plant Disease Vol. 21 No. 2 99.78% 유사도(EZ-taxon blasting 결과)를 보였다(Fig. 2C).

5 54 Research in Plant Disease Vol. 21 No % 유사도(EZ-taxon blasting 결과)를 보였다(Fig. 2C). 이에 상기에서 분리된 균주를 Streptomyces netropsis AN110065라 명명하였으며, 2013년 09월 23일 자로 국제 기탁기관에 기탁하 여 KCTC12490BP라는 기탁 번호를 부여받았다. Fig. 3. Nematicidal activities of two organic layers and aqueous layer obtained from the methanol extract of Streptomyces netropsis AN fermentation broth against second-stage juveniles of Meloidogyne incognita. The mortalities were measured 72 h after treatment. Each value represents the mean ± standard deviation of three replicates. Bars indicate significant differences at 5% probability level. 용매 분획별 살선충 활성. AN 배양액의 메탄올 추 출액의 에틸 아세테이트 추출물이 1,000 mg/ml 농도에서 83.5% 수준으로 가장 높은 살선충 활성을 나타내었다(Fig. 3). 부탄올 추출물의 경우 1,000 mg/ml 농도에서 살선충 활성을 보이지 않 았으며, 물 추출물의 경우 약 23.4%의 낮은 살선충 활성을 보였 다(Fig. 3). 균주 배양액의 뿌리혹선충에 대한 포트 실험. AN 배양액의 10배, 100배, 1,000배 희석액 처리구에서 각각 , Fig. 4. Inhibition activity of the fermentation broth of Streptomyces netropsis AN on the gall formation on tomato plant roots caused by Meloidogyne incognita (A) and the treated plants at 6 weeks after infection of root-knot nematodes (B). Gall index: 0 = 0-10%, 1 = 11-20%, 2 = 21-50%, 3 = 51-80%, 4 = 81-90%, and 5 = % of roots galled (Taylor and Sasser, 1978). Values are mean ± SD of five replicates and means were separated by Duncan s multiple range test (P = 0.05) 김진철o.indd 오후 2:38:56

6 Research in Plant Disease Vol. 21 No , 4.0 (Fig. 4)., AN , 100 1,000 (Fig. 4). 0.2 (Fig. 4). ( ) %, (M. incognita) (Qiao, 2011). S. netropsis AN , 40%,. Wei (2014) Bacillus subtilis jdm2 (10 9 cfu/ml) 39%,, Kim (2011) Streptomyces samposonii KK1024,,. Kim (2011) S. sampsonii KK % 21%, 20% 47%, Wei (2014) B. subtilis jdm2 67% 24 64% 72 77%.. S. netropsis AN AN110065, cell cell. Kakar (2014) in vivo (bacterial brown stripe) Brevibacillus laterosporus B4 cell (intracellular substance). avermectin Streptomyces avermiltiles KCTC 9698, 10 75%. in vitro, ( )., (intracellular substance) (extracellular substrance). avermectins, bafilomycin, lecanicidins, 3-methoxy-2-methyl-carbazole-1,4-quinone, carbazomycins D F fervenulin (Burg, 1979; Lacey, 1995; Nonaka, 2000; Ruanpanun, 2010; Ruanpanun, 2011). S. netropsis AN S. netropsis AN110065,. (M. incognita).,,,. methyl bromide.. 2,700 50%. AN % 50% 20% %, %. 16S rrna sequencing S. netropsis 99.78%. S. netropsis AN110065,, 1,000 mg/ml 83.5%. AN

7 56 Research in Plant Disease Vol. 21 No. 2 AN Acknowledgement This study was performed with support from the Cooperative Research Program for Agricultural Science and Technology Development (Project PJ ), Rural Development Administration, Republic of Korea. This research was supported by a grant ( ) from the R&D Program of MOTIE/KEIT of Republic Korea. References Abbott, W. S A method for computing the effectiveness of an insecticide. J. Eco. Entomol. 18: Barker, K. R., Schmitt, D. P. and Imbriani, J. L Nematode population dynamics with emphasis on determining damage potential to crops. In: K.R. Barker, C. C, Carter, and J. N Sasser (eds.). An advanced treatise on Meloidogyne, Vol. II. pp North Carolina State University, Raleigh, North Carolina, USA. Berdy, J Bioactive microbial metabolites. J. Antibiot. 58: Burg, R. W., Miller, B. M., Baker, E. E., Birnbaum, J., Currie, S. A., Hartman, R., Kong, Y. -L., Monaghan, R. L., Olson, G., Putter, I., Tunac, J. B., Wallick, H., Stapley, E. O., Oiwa, R. and Omura, S Avermectins, new family of potent anthelmintic agents: producing organism and fermentation. Antimicrob. Agents Chemother. 15: Caboni, P., Aissani, N., Cabras, T., Falqui, A., Marotta, R., Liori, B., Ntalli, N., Sarais, G., Sasanelli, N. and Tocco, G Potent nematicidal acitivity of phthalaldehyde, salicylaldehyde, and cinnamic aldehyde against Meloidogyne incognita. J. Agric. Food Chem. 61: Cabrera, J. M., Menjivar, R. D., Dababat, A. A. and Sikora, R. A Properties and nematicide performance of avermectins. J. Phytopathol. 161: Cho, M. R., Lee, B. C., Kim, D. S., Jeon, H. Y., Yiem, M. S. and Lee, J. O Distribution of plant-parasitic nematodes in fruit vegetable production areas in Korea and identification of root-knot nematodes by enzyme phenotypes. Korean J. Appl. Entomol. 39: Choi, Y. E. and Choo, H. Y A study on root-knot nematodes affecting economic crops in Korea. Korean J. Plant Prot. 17: Hesseltine, C. W., Benedict, R. G. and Pridham, T. G Useful criteria for species differentiation in the genus Streptomyces. Ann. N. Y. Acad. Sci. 60: Hwang, S. M., Park, M. S., Kim, J. C., Jang, K. S., Choi, Y. H. and Choi, G. J Occurrence of Meloidogyne incognita infecting resistant cultivars and development of an efficient screening method for resistant tomato to the Mi-virulent nematode. Korean J. Hort. Sci. Technol. 32: Kakar, K. U., Nawaz, Z., Cui, Z., Almoneafy, A. A., Zhu, B. and Xie, G. L. 2014, Characterizing the mode of action of Brevibacillus laterosporus B4 for control of bacterial brown strip of rice caused by A. avenae subsp. avenae RS-1. Wold J. Microbiol. Biotechnol. 30: Khalil, M. S Abamectin and azadirachitin as eco-friendly promising biorational tools in integrated nematodes management programs. J. Plant Pathol. Microb. 4: Khan, Z., Kim, S. G., Jeon, Y. H., Khan, H. U., Son, S. H. and Kim, Y. H A plant growth promoting rhizobacterium, Paenibacillus polymyxa strain GBR-1, suppresses root-knot nematode. Bioresource Tech. 99: Kim, D. G Occurrence of root-knot nematodes on fruit vegetables under greenhouse conditions in Korea. Res. Plant Dis. 7: (In Korean) Kim, K. H., Joe, Y. A., Choi, S. R. and Goo, Y. M Comparative studies on streptomycin producing strains and media. Korean J. Biotechnol. Bioeng. 4: Kim, S. S., Kang, S. I., Kim, J. S., Lee, Y. S., Hong, S. H., Naing, K. W. and Kim, K. Y Biological control of root-knot nematode by Streptomyces sampsonii KK1024. Korean J. Soil Sci. Fert. 44: Lacey, E., Gill, J. H., Power, M. L., Rickards, R. W., O shea, M. G. and Rothschild, J. M Bafilolides, potent inhibitors of the motility and development of the free-living stages of parasitic nematodes. Int. J. Parasitol. 25: Lane, D. J S/23S rrna sequencing. In: Stackebrandt E, Goodfellow M. editors. Nucleic acid techniques in bacterial systematics. New York, NY: John Wiley & Sons, Inc. pp Le Dang, Q., Kim, W. K., Nguyen, C. M., Choi, Y. H., Choi, G. J., Jang, K. S., Park, M. S., Lim, C. H., Luu, N. H. and Kim, J.-C Nematicidal and antifungal activities of annonaceous acetogenins from Annona squamosa against various plant pathogens. J. Agric. Food Chem. 59: McCart, J. P Molecular approaches toward resistance to plantparasitic nematdoes. In Cell Biology of Plant Nematode Parasitism, Plant Cell Monogr 15th ed. Berg, R.H and C.G. Tayor. (Eds). pp Springer. St. Louis, USA. Nonaka, K., Tsukiyama, T., Okamoto, Y., Sato, K., Kumasaka, C., Yammoto, T., Maruyama, F., and Yoshikaw, H New milbemycins from Streptomyces hygroscopicus subsp. aureolacrimosus: fermentation, isolation and structure elucidation. J. Antibiot. 53: Oka, Y., Nacar, S., Putievsky, E., Ravid, U., Yaniv, Z. and Spiegel, Y Nematicidal activity of essential oils and their components against the root-knot nematode. Phytopathology 90: Park, J., Seo, Y. and Kim, Y. H Biological control of Meloidogyne hapla using an antagonistic bacterium. Plant Pathol. J. 30: Qiao, K., Liu, X., Wang, H., Xia, X., Ji, X. and Wang, K Effect of

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