식물병연구 Research Article Open Access Res. Plant Dis. 24(3): 213-220 (2018) https://doi.org/10.5423/rpd.2018.24.3.213 Botrytis cinerea Colletotrichum acutatum Sorangium cellulosum Subspecies Classifying and Characterizing the Two Groups of Antagonistic Sorangium cellulosum against Botrytis cinerea and Colletotrichum acutatum *Corresponding author Tel: +82-41-530-2282 Fax: +82-41-530-2393 E-mail: scyun@sunmoon.ac.kr ORCID https://orcid.org/0000-0001-6295-8642 * Tae-Hoon Koo and Sung-Chul Yun * Department of Pharmaceutical Engineering and Biotechnology, Sunmoon University, Asan 31460, Korea Received April 30, 2018 Revised May 9, 2018 Accepted May 9, 2018 We classified the previously reported antagonistic strains of Sorangium cellulosum into 5 subspecies (A-E). Four strains were antagonistic to Botrytis cinerea (AB group) and two strains were antagonistic to Colletotrichum acutatum (AC group). According to the genetic and sequential analyses with standard genes, xynb1, bgla2, groel1 for grouping, all strains of AB group were belonged to subspecies C and all strains of AC group were belonged to subspecies D. In addition, high pressure liquid chromatography with the culture filtrates confirmed the genetic results, because AB group had peaks with retention time at 20-22.5 minutes, whereas AC group had no peak. There was positive relationship (R 2 =0.9652) between the control values of infecting B. cinerea on cherry tomatoes and the main peak areas of chromatograms among the four isolates of AB group. From the subspecies results of AB group, the main peak of KYC 3270 was expected to be epothilone D. However the retention times of the standard of commercial epothilone D and the main peak of KYC 3270 culture filtrate were different as 9.9 and 11.581 min., respectively. Finally, the antagonistic metabolite of AB group was inferred as 7-ketone epothilone D. Keywords: 7-ketone epothilone D, Antagonistic metabolites, Chromatography, Myxobacteria, Phytopathogenic fungi Sorangium cellulosum Research in Plant Disease pissn 1598-2262, eissn 2233-9191 www.online-rpd.org. Kim Yun (2011) Botrytis cinerea, Colletotrichum acutatum, Pyricularia grisea, Penicillium sp. S. cellulosum. S. cellulosum 318 C. acuatum 29 23 P. grisea Penicillin sp.., B. cinerea S. cellulosum The Korean Society of Plant Pathology This is an open access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
214 Research in Plant Disease Vol. 24 No. 3 79 2.7, 7 B. cinerea C. acutatum. AB (Antagoistic to B. cinerea; AB group) 79 AC (Antagoistic to C. acutatum; AC group) 29 7. AB 79 B. cinerea KYC 3130, 3247, 3248, 3270 (Kim Yun, 2011), AC 29 KYC 3262, 3279 KYC 3262 2011 3 (Yun, 2014). 5 (subspecies A-E) (Lee, 2013). groel1, xynb1, bgla2 (Jiang, 2008), groel1 A, xynb1 E, B, C, D. B, C, D groel1, xynb1. xynb1 endo-1,4-b-glucanase, cellulose, lichenin cereal--d-glucan 1,4- -glucoside (Coughlan Hazlewood, 1993). bgla2 -glucosidase cellulase. groel1 (biofilm) mycolic acid (Ojha, 2005). (HPLC). Lee (2013) 5 (profile), (retention time, rt)., A A, B B1/ B2/B3, C C, E E1/E2, D (Lee, 2013)., S. cellulosum, A disorazole, C epothilone spirangien, D ambruticin, E soraphen (Lee, 2013)., 5 2 (Lee, 2013)., S. cellulosum ( ) (). S. cellulosum, 1 (standard). AB AC S. cellulosum. HPLC. AB B. cinerea, HPLC epothilone D AB.. B. cinereal (No. 43528) (Korean Agricultural Culture Collection: KACC), C. acuatum SWS 2000. 25 o C 7 110 5 conidia/ml. S. cellulosum (KYC 3130, 3247, 3248, 3270 AB, KYC 3262, 3279 AC ) (Myxobank) -70 o C. ST21 (0.5 cm2 cm) 7. CSG (Casitone, Soluble starch, Glucose ) 100 ml (Kim Yun, 2011). HPLC ST21 CYS (Casitone, Yeast extract, Soluble starch ) 100 ml (Lee, 2013).
Research in Plant Disease Vol. 24 No. 3 215 S. cellulusom PCR. 6 DNA, PCR Lee (2013). Macrogen. A KYC 3064, B KYC 3234, C KYC 3013, D KYC 3156, E KYC 3076. National Center for Biotechnology Information (NCBI). Mega6 program. HPLC. 100 ml CYS 180 rpm, 32 o C 5,., 1-2 ml. pore size 0.2 mm 100. (Incidence). 4, 10, 4. (control value) (x, y),. 2 epothilone HPLC. epothilone D (PMID 268909, Sigma-Aldrich, USA) 1 ml 1,000 zorbax C-18 HPLC. 2 15 A B (Kern, 2015). A acetonitrile 8 2, B acetonitrile. 0-8 B 20% 90%, 8-15 90%. AB, AC KYC 3270 KYC 3262. HPLC. 1 ml Lee (2013) S. cellulosum (solvent system). (Shimazu HPLC 2300, Japan) Agilent zorbax C-18 (Agilent, USA) 1 50., 1 ml.. PCR xynb1, bgla2, groel1, AB AC 6 (Fig. 1). AB AC B, C, D.. B. cinerea Kim Yun (2011) KYC 3270. 24 70%, 10 ml, positive control fludioxonil(, Syngenta, China, a.i. 20%). 24. 25 o C 24 5-7 Fig. 1. Electrophoresis of Sorengium cellulosum PCR products of xynb1 gene (lane 1, 4, 7, 10, 13, 16), bgla2 gene (lane 2, 5, 8, 11, 14, 17) and groel1 gene (lane 3, 6, 9, 12, 15, 18). The three genes are for subspecies S. cellulosum. KYC 3130, 3247, 3248, and 3270 were AB (Antagonistic to Botrytis cinerea) strains and KYC 3262, and 3279 were AC (Antagonistic to Colletotrichum acutatum) strains of S. cellulosum. Lane M1 and M2 were 1 kb ladder maker.
216 Research in Plant Disease Vol. 24 No. 3 Fig. 2. Phylogenetic tree of the eight strains of Sorengium cellulosum PCR products of xynb1 (A) and groel1 (B) genes. The underlined KYC 3064, 3234, 3013, 3156, and 3076 were the type strains of subspecies A, B, C, D, and E, respectively. Fig. 3. Chromatograph of Sorengium cellulosum extracts of AB (Antagonistic to Botrytis cineria) group isolates (KYC 3130, 3247, 3248, and 3270) with HPLC solvent system I for subspecies grouping. The arrows were main peaks at 20-22.5 min. retention time. 한 아종 분류를 위하여 염기서열 분석을 실시하여 NCBI에 등 록된 각 아종 내 대표 균주들의 xynb1, groel1 유전자의 유사성 을 비교한 결과, AB 계열인 KYC 3130, KYC 3247, KYC 3248, KYC 3270 균주들은 아종 C의 대표 균주인 KYC 3013과 유사한 반면, AC 계열인 KYC 3262와 KYC 3279는 아종 D의 대표 균주인 KYC 3156과 유사하였다(Fig. 2). PCR과 염기서열 분석 결과, AB 계열 은 아종 C로, AC 계열은 아종 D로 분류하였다. 분석화학적 아종 분류. 아종 분류를 위해 HPLC 크로마토 그램을 분석한 결과, AB 계열인 KYC 3130, 3247, 3248, 3270은 머무름시간 20-22.5분 사이에 반복적인 특성 피크가 존재하였 다(Fig. 3). 이 피크는 UV 254 nm에서 나타나 epothilone로 추 론하였다. 그런데, 같은 방법으로 Lee 등(2013)이 분석한 아종 분류를 위한 HPLC 크로마토그래피 결과 아종 C의 특성 피크 머무름시간은 28-29분이어서 본 연구결과인 AB 계열 특성 피 크의 머무름시간 20-22.5분과 5분 이상의 머무름시간 차이를 보이는데, 이는 기계적 차이로 볼 수 없으므로 AB 계열의 특성 피크는 epothilone라기보다는 유도체 중 하나인 epothilone D 로 판단하고, 이를 동정하기 위한 2차 HPLC 실험을 준비하였 다. 한편, AC 계열인 KYC 3262와 3279은 20-22.5분에 피크 가 간혹 있었지만, 반복된 실험에서 지속적으로 검출되지 않 Fig. 4. Chromatograph of Sorengium cellulosum extracts of AC (Antagonistic to Colletotrichum acutatum) group isolates (KYC 3248 and 3279) by HPLC solvent system I for subspecies grouping. The main peak was not detected on both isolates. 아(Fig. 4), 특성 피크가 없는 아종 D의 특징(Lee 등, 2013)을 보였다. 따라서 HPLC 분석화학적 아종 분류 결과와 유전학적 아종 분류 결과들 모두 AB 계열은 아종 C, AC 계열은 아종 D 로 일치하였다. 특징 피크 면적값과 방제가의 상관관계. AB 계열에 속하 는 4가지 균주들의 배양 여액을 방울토마토(Fig. 5)와 딸기(Fig.
Research in Plant Disease Vol. 24 No. 3 217 Fig. 5. Control values of AB group (KYC 3130, 3247, 3248 and 3270) of Sorengium cellulosum with infection Botrytis cinerea on cherry tomato (A). Control values were calculated the occurrence of gray molds among 4 fruits of cherry tomato. Correlation between control values of AB strains of S. cellulosum and relative area (%) of the main peak on HPLC chromatograph by solvent system I. The four strains of AB group are KYC 3130 (-), 3247 ( ), 3248 ( ) and 3270 ( ). Means with the same letters are not significantly different (p<0.05). Data were the average of four replications. Fig. 6. Control values of AB group (KYC 3130, 3247, 3248 and 3270) of Sorengium cellulosum with infection Botrytis cinerea on strawberry (A). Control values were calculated the occurence of gray molds among 10 fruits of strawberry. Correlation between control values of AB strains of S. cellulosum and relative area (%) of the main peak on HPLC chromatograph by solvent system I. The four strains of AB group are KYC 3130 (-), 3247 ( ), 3248 ( ) and 3270 ( ). Means with the same letters are not significantly different (p<0.05). Data were the average of four replications. 6). AB KYC 3247, 3248, 3270 75.0%, 75.0%, 100%, positive control fludioxonil 50%. KYC 3130 12.5%(Fig. 5A)., fludioxonil 87.5%, KYC 3130, 3247, 3248 4.2%, 16.7%, 16.7%. KYC 3270 79.2% fludioxonil (Fig. 6A). AB 3.631 (KYC 3130), 8.432 (KYC 3247), 7.264 (KYC 3248), 9.926 (KYC 3270)., R 2 =0.9652 (Fig. 5B), R 2 =0.5891 (Fig. 6B), KYC 3247 KYC 3248
218 Research in Plant Disease Vol. 24 No. 3 11.582, Fig. 7C AC KYC 3262 8-10, epothilone D (Fig. 7B, C). Fig. 7B KYC 3270 11.582 Kern (2015) 10.9 7-ketone epothilone AB KYC 3270. Fig. 7. Chromatographs of epothilone D stadard (A), KYC 3270 (B), and KYC 3262 (C) extracts by HPLC solvent system II. The arrows had main peak at 11.582 min.retention time.. HPLC. epothilone D AB KYC 3270 AC KYC 3262 HPLC. epothilone D (Fig. 7A) 9.310(Fig. 7A), Kern (2015) epothilone D 9 9.310 epothilone D,. Fig. 7B AB KYC 3270, AB 4 S. cellulosum C, AC 2 S. cellulosum D,. AB, AC S. cellulosum xynb1, bgla2, groel1. HPLC AB 20-22.5, AC. AB (R 2 =0.9652). AB, AC. C spirangien epothilone, epothilone epoa spirangien spig (Lee, 2013). spirangien A-G, epothilone A-K (Frank, 2007; Kern, 2015). Frank (2007) Niggemann (2005) spirangien A 23.6, UV maxima 303, 318, 333, 351 nm. UV 254 nm epothilone epothilone D 1. Epothilone UV 254 nm, epothilone B 6.9, 14-OH epothilone D 5.5, epothione D 9 (Kern, 2015). Rogalska (2013) Wang (2009) epothilone A-D,
Research in Plant Disease Vol. 24 No. 3 219., (Inhibition Concentration, IC) 1.57 g/ml (epothilone A ), 0.00249 g/ml (epothilone B), 0.846 g/ ml (epothilone B ), epothilone C D. AB 7-ketone epothilone D epothilone D (Kern, 2015). HPLC 7-ketone epothilone D (liquid chromatography with mass spectrometry, LC-MS) LC-MS (supplementary data). 7-ketone epothilone D (Kern, 2015)., H 2 O, m/z 472, CO2 m/z 402, 7 162. epothilone D epothilone B 7-ketone epothilone D (Kern, 2015). AC KYC 3262 2011-2013 330.6 m 2 3 (Yun, 2014). KYC 3262 3 31%, 89%, 82%, dithianon 19%, 97%, 91% 10% (Yun, 2014)., HPLC AC, D ambruticin (Lee, 2013), AC ambruticin. AB S. cellulosum 4 KYC 3130, 3247, 3248, 3270 C 7-ketone epothilone D. 318 79 72 C., Ringel (1997) D ambruticin 0.78 mcg/ml B. cinerea. B. cinerea S. cellulosum. Kim Yun (2011) 318 S. cellulosum B. cinerea-c. actatum 7 S. cellulosum ambruticin., epothilone ambruticin, 7 C. acutatum 22. Sorangium cellulosum Botrytis cinerea 4(Antagonistic to Botrytis, AB ) Colletotrichum acutatum 2(Antagonistic to Colletotrichum, AC ) 5(A-E) S. cellulosum (subspecies). xynb1, bgla2, groel1 AB C, AC D., (HPLC) AB 4 20-22.5, AC 2 AB C, AC D. AB 4 B. cinerea, (R 2 =0.9652). AB C epothilone D, HPLC epothilone D 9.9, KYC 3270 11.581. AB epothilone 7-ketone epothilone D. Conflicts of Interest No potential conflict of interest relevant to this article was reported. Acknowledgments This work was supported by Sun Moon University Research Grant of 2018.
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