식물병연구 Research Article Open Access Res. Plant Dis. 21(2) : 50-57(2015) http://dx.doi.org/10.5423/rpd.2015.21.2.050 Meloidogyne incognita Streptomyces netropsis Characterization of Streptomyces netropsis Showing a Nematicidal Activity against Meloidogyne incognita 1,2 3 4 3 3 3 4 2 5 1 * 1, 2, 3, 4, 5 *Corresponding author Tel : +82-62-530-2132 Fax: +82-62-530-2139 E-mail: 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 500-757, Korea 2 Department of Plant Medicine, Chungbuk National University, Cheongju 361-763, Korea 3 Eco-friendly New Materials Research Group, Korea Research Institute of Chemical Technology, Daejeon 305-600, Korea 4 Microbial Resource Center, Korea Research Institute Bioscience and Biotechnology, Daejeon 305-806, Korea 5 World Institute of Kimchi, an Annex of Korea Food Research Institute 675-1, Imamdong, Namgu, Gwangju 503-360, 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 AN110065 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 AN110065 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 AN110065 did not show any phytotoxicity. The results suggest that S. netropsis AN110065 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 1598-2262, eissn 2233-9191 This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Research in Plant Disease Vol. 21 No. 2 51 (Meloidogyne spp.). 11%, 1 180 (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).. 60-70% (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) 5 100 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. AN110065 Bennett 28 o C 14. (Scanning electron microphotograph, SEM, JSM-6700F, JEOL Ltd, Japan). AN110065 glutaraldehyde(2.5%, 0.1 M phosphate buffer, ph 7.0),. isoamyl acetate, CO 2 isoamyl acetate,. 16S rrna. AN110065 5 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
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 1000. 뿌리혹선충의증식및유충분리. Hwang (2014) ( ) (255 o C). (, ) 9.5 cm, 3. 10,000, 45 60.,. Barker (1985), 1 cm 0.5% (sodium hypochlorite) 60. 230 mesh 550 mesh. 550 mesh 3, Baermann funnel (Southey, 1986). 방선균의살선충활성스크리닝. 2,700 96-well tissue culture plate(becton Dickinson, Franklin Lakes, NJ). 1 ml 20. 4 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% 25-27 o C. 3, 24 72 (Olympus CKX41, Tokyo, Japan), Abbotts formula (Abbott, 1925). (%) = [( - ) / (100 - )] 100. AN110065 균주용매분획의살선충활성. AN110065 GSS 28 o C, 150 rpm 10. 500 ml, Whatman No 1.. 50 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, 24 72. 토마토포트에서뿌리혹선충병방제활성검정. AN110065. 5 (253 o C). 9.5 cm 400 g 2. 10,000. GSS 28 o C, 150 rpm 10 AN110065 10, 100, 1,000 4. (a. i. Fosthiazate 5%) 2,000 1,. 5, 6 Taylor Sasser(1978). 0-5 6 (0, 0%; 1, 1-20%; 2, 21-40%, 3, 41-60%, 4, 61-80%, 5, 81-100%). 통계분석. SAS(SAS Institute, Inc, 1989, Cary, NC) (one-way analysis of variance)
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 AN110065 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. AN110065 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 AN110065 균주의 배양여액만을 처리했을 때는 활성을 보이지 않았다. 무 처리 대조구로 멸균수:메탄올(1:1, v/v) 용액만을 20% 처리 했을 때 치사활성은 없었다. AN110065 균주의 형태학적 특성 및 16S rrna sequencing 분석. AN110065 균주는 흰색의 포자를 형성하였으며, 주사 전자현미경으로 관찰한 결과 rectiflexible 한 포자체인 형태를 보이고 원통형 모양의 0.4-1.2 mm 크기의 포자를 형성하였다 (Fig. 2A와 B). 16S rrna 유전자 염기서열에 기초한 분자 계통학적 분석 결 과, AN110065 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 AN110065 on Bennett s agar at 28oC for 14 days. Neighbour-joining phylogenetic tree based on 16S rrna gene sequences showing the relationship between strain AN110065 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. 50-57 0078-김진철o.indd 53 2015-06-23 오후 2:38:56
54 Research in Plant Disease Vol. 21 No. 2 99.78% 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 AN110065 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. AN110065 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. AN110065 배양액의 10배, 100배, 1,000배 희석액 처리구에서 각각 2.4 2.8, Fig. 4. Inhibition activity of the fermentation broth of Streptomyces netropsis AN110065 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 = 91-100% 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). 50-57 0078-김진철o.indd 54 2015-06-23 오후 2:38:56
Research in Plant Disease Vol. 21 No. 2 55 3.2, 4.0 (Fig. 4)., AN110065 10, 100 1,000 (Fig. 4). 0.2 (Fig. 4). ( ). 40-46%, (M. incognita) (Qiao, 2011). S. netropsis AN110065 10, 40%,. Wei (2014) Bacillus subtilis jdm2 (10 9 cfu/ml) 39%,, Kim (2011) Streptomyces samposonii KK1024,,. Kim (2011) S. sampsonii KK1024 10% 21%, 20% 47%, Wei (2014) B. subtilis jdm2 67% 24 64% 72 77%.. S. netropsis AN110065. 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 AN110065.. S. netropsis AN110065,. (M. incognita).,,,. methyl bromide.. 2,700 50%. AN110065 10% 50% 20% 1 78.9%, 3 94.1%. 16S rrna sequencing S. netropsis 99.78%. S. netropsis AN110065,, 1,000 mg/ml 83.5%. AN110065 10.
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