: P. flourescensb. subtilis E. herbicola Eh252E. carotovora subsp. betavasculorum Ecb168, E. carotovora subsp. carotovora [27] Lysobacter,, 4

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1 微生物学通报 JUL 20, 2009, 36(7): 974~980 Microbiology 研究报告 2009 by Institute of Microbiology, CAS 一种新型生防细菌菌株 13-1 鉴定及其生物学特性 1* 姬广海 2 魏兰芳 1 吴亚鹏 ( ) ( ) 摘要 : 13-1 是从魔芋根际土壤中筛选得到的一株革兰氏阴性生防细菌, 抑菌试验结果表明它对 7 种植物病原细菌和 8 种病原真菌具有拮抗作用形态和常规生理生化特征鉴定, 发现该菌株与溶杆菌属 Lysobacter 特征很相近 Biolog 鉴定和 16S rdna 基因序列分析表明, 菌株 13-1 与已报道的抗生素溶杆菌 Lysobacter antibioticus DSM 2044(=ATCC29479) 同源性高达 99% 二者在所建系统发育树中处于同一分枝, 将 13-1 菌株鉴定为抗生素溶杆菌 (L. antibioticus) 关键词 : Biolog 鉴定, 16S rdna 测序, 抗生素溶杆菌 (L. antibioticus) Identification and Biological Characteristics on a Novel Strain 13-1 from Rhizosphere of Amorphophallus konjac JI Guang-Hai 1* WEI Lan-Fang 2 WU Ya-Peng 1 (1. National Center for Agro-biodiversity, Yunnan Agricultural University, Kunming, Yunnan , China) (2. Experimental Agriculture Center, Yunnan Agricultural University, Kunming, Yunnan , China) Abstract: Strain 13-1 was gram negative biocontrol bacterial strains isolated from Amorphophallus konjac. In vitro antagonistic assay showed that strain could suppress the growth of seven plant bacterial pathogens and eight pant fungal pathogens. The results of phenotypic and general physiological and biochemical properties showed that strain 13-1 homologized with which descripted for genus Lysobcter. 16S rdna determination and analysis was used for further identification, which showed that 16S rdna sequence of strain 13-1 shared 99% homologies with published sequence of L.antibioticus DSM2044 from GenBank, and both sequences constituted a branch in phylogenetic tree. Based on these results, it is considered that the strain 13-1 belongs to one strain of L.antibioticus. Keywords: Identification, Biocontrol strain 13-1, 16S rdna, L. antibioticus, [1], 1980, 1 200,, 15%~20%, 100% (),,, 基金项目 :(No ); ()(No. nyhyzx07-056) * 通讯作者 : : jghai001@yahoo.com.cn 收稿日期 : ; 接受日期 :

2 : P. flourescensb. subtilis E. herbicola Eh252E. carotovora subsp. betavasculorum Ecb168, E. carotovora subsp. carotovora [27] Lysobacter,, 4, L. enzymogenes L. antibioticus L. brunescens L. gummosus [8],, L. koreensis L. daejeonensis L. yangpyeon gensisl. niabensis [9], C3,, [10] 3.1T8N4-7Lysobacter sp. XL1 Lysobacter sp. SB-K88 L. lactamgenus YK90, [1115] ; [16],,,,,, 1 材料与方法 1.1 供试菌株 , 1.2 平板抑菌作用测定 13-1 PDA 5 mm, 2.5 cm, 3, 2, 28 C 5 d CFU/mL, NA, 13-1, 4, 2, 28 C 2 d~3 d, 1.3 培养性状和菌体形态特征观察 13-1 NAR 2 A, (KOH),, 18 hpta, EM-1200EX, : 48 h,, [17] 13-1 [18] 1.4 Biolog 微生物自动鉴定仪鉴定 : 13-1, BUG, 28 C 20 h~24 h (Biolog ),, :, 61%±2% 8 Biolog GN 150 μl, 96, 30 C, 4 h 24 h,, S rdna 的序列测定及其系统发育学分析 DNA, 13-1 DNA, 27 f (5 -AGAGTTTGATCCT GGCTCAG-3 ) 1492 r (5 -GGCTACCTIGTIACGA CTT-3 ) PCR PCR (50 μl): 10 5 μl, dntps (2.5 mmol/ml) 4 μl, 27 f (10 μmol/ml)2 μl, 1942 r (10 μmol/ml) 2 μl, DNA 2 μl, DNA(5 U/µL) 0.25 μl, ddh 2 O μlpcr: 95 C 3 min; 94 C 1 min, 55 C 1 min, 72 C 1.5 min, 30 ; 72 C 8 min,, PCR,, ABI PRISM 377~96, 27 f 1492 r

3 976 微生物学通报 2009, Vol.36, No.7 Blast GenBank S rdna 序列分析和系统发育树的构建 GenBank, Blastn ( blast), GenBank 16S rdna, Clustal X, (Neighbor-joining method), bootstrap 1000,, 2 2 结果与分析 2.1 菌株平板抑菌作用 ( 1), 13-1 My9ECC2WM2, 11.4 mm~13.0 mm,, 培养性状及形态特征观察 NA,,,,, Table 1 表 1 生防细菌 13-1 对不同病原菌的抑菌圈直径 Inhibition zone diameter of bio-control bacteria strain 13-1 against various pathogens () Plant bacterial(fungal) pathogen Pectobacterium carotovorum ssp. carotovora Xanthomonas oryzae pv. oryzae Ralstonia solanacearum P. syringae pv. tabaci P. syringae pv. tabaci Xathomonas oryzae pv. oryzicola Xanthomonas axonopodis pv. dieffenbachiae Xanthomonas campestris pv. campestris Pyricularia grisea (Cooke) Sacc. Strain code My9 ECC2 Wm2 (mm) Diameter of inhibition zone Tb23 CX10 T5 Rs68 XCD-S Y Host plants Konnyaku Cabbage Zantedeschia Rice Various plants Tobacco Orientia tobacco Rice Anthurium Cabbage Rice Pythium sp. SQ Panax notoginseng Fusarium orysporum Schlf. sp. Vanillae (Tucker) Gordon F. solani (Mart.) Appel & Wollenw. emend Snyd. & Hans Exserohilum turcicum (pass.) Leonard & Suggs Bipolaris maydis (Nishik & Miyabe) shoem Phytophthora nicotianae Alternaria alternate (Fries) Keissler F1 LH5 DB XB , CX Vanilla Aloe barbadensis Maize Maize Tobacco Tobacco

: 13-1 977 图 1 菌株 13-1 菌落形态 Fig. 1 Colony of strain 13-1 : A: ; B:. Note: A: Early time; B: Later time. (0.6~0.9)μm (1.5~ 2.0)μm 2.3 常规生理生化特征鉴定 2, (Xanthomonas axonopodis pv.

4 : 图 1 菌株 13-1 菌落形态 Fig. 1 Colony of strain 13-1 : A: ; B:. Note: A: Early time; B: Later time. (0.6~0.9)μm (1.5~ 2.0)μm 2.3 常规生理生化特征鉴定 2, (Xanthomonas axonopodis pv. dieffenbachiae X CD ),, H 2 S,,,, V.P, 80,,, 2% NaCl, 5% NaCl,, 2.4 Biolog 微生物鉴定系统鉴定 Test items Gram stain Aerobic growth Anaerobic growth H 2 SH 2 S Produce Catalase Oxidase Glucose acidification Urease Arginine dihydrolase Nitrate deoxidization V.P Voges-Proskauer test Hydrolysis of starch Casein hydrolysis Hydrolysis of gelatin : +: ; :. Note: +: Positive; : Negative. Table 2 表 2 菌株的生理生化性状 Physiological and biochemical characteristers of bacterial strains Strain test 13-1 X CD Test items Levan formation NaCl 2% Growth on 2% NaCl NaCl 5% Growth on 5% NaCl NaCl 10% Growth on 10% NaCl Frutcose Arabinose Glucose Histidine Mannose L-Proline D-Galactose Sucrose D- D-Xylose Trehalose Cellobiose Strain test 13-1 X CD + N

5 978 微生物学通报 2009, Vol.36, No.7 Biolog, 24 h,, N--D-N--D- D-α-D-D-3- α-α- L- L-; L-L- Glycogen Maltoseα-D- L. antibioticus DSM 2044 Biolog 13-1, 50%, Stenotrophomonas maltophilia, 46.1% S rdna 的序列测定及其系统发育学分析生防细菌 S rdna 的序列测定 : 27 f 1492 r S rdna PCR, 1.5 kb PCR, S rdna 1268 bp, GenBank DQ 系统发育学分析 : GenBank, 13-1 L. antibioticus DSM 2044(ATCC29479) 99%, N-J ( 2),, 13-1 L. antibioticus OC5DSM 2044(ATCC29479),, 91% ( 2), 13-1 (L. antibioticus 13-1): (Xanthomonadaceae), (Lysobacter), (L. antibioticus) 3 讨论 13-1, NA 图 2 生防菌 13-1 的系统发育树 Fig. 2 Phylogenetic tree constructed based on strain S rdna gene sequence : bootstrap1000 ; GenBank 16S rdna. Note: The number at each branch points is percentage supported by bootstrap; Strain No. and GenBank accession number.

6 : ,, [20], L. antibioticus ATCC29479 D-, 13-1 Biolog, Biolog microstation,,,, Biolog 13-1, Stenotrophomonas (SIM 0.46), ( 16 h~24 h SIM0.5) Biolog,, 16S rdna, 16S rdna 98%, ; 93%~95%, [20], 13-1 L. antibioticus DSM 2044(ATCC29479) 99%, 13-1, L. antibioticus 13-1,,,, L. antibioticus 13-1, 参考文献 [1] Perombelon MCM, Kelman A. Blackleg and other potato disease caused by soft Erwinia proposal for revision of terminology. Plant Dis, 1987, 71(3): [2] Costa JM, Joyce EL. Derivation of Mutants of Erwinia carotovora subsp. betavasculorum deficient in export of pectolytic enzymes with potential for biological control of potato soft rot. Applied and Enviromental Microbiology, 1994, 60(7): [3] Folman LB, De Klein MJEM, Postma J, et al. Characterization of Lysobacter enzymogenes (Christensen and Cook, 1978) strain 3.1T8, a powerful antagonist of fungal disease of cucumber. Microbiology Research, 2003, 158(2): [4] Hendawy HE, Zeid IM, Mohamed, et al. The biological control of soft rot disease in melon caused by Erwinia carotovora subsp. carotovora using Pseudomonas fluorescens. Microbiological research, 1998, 153(11): [5] Seo ST, Furuya S, Iiyama K, et al. Characterization of an antibacterial substance produced by Erwinia carotovora subsp. carotovora Ecc 32. Journal of General plant pathology, 2004, 70(5): [6] Vanneste JL, Yu J, Cornish DA. Biological Control of bacterial soft rot. ISHS Acta Horticulturae 96: International Postharvest Science Conference Postharvest, 1996, p.464. [7] Zamanian S, Shahidi GH, Saadoun I. First report of antibacterial properties of a new strain of Streptomyces plicatus(strain 101) against Erwinia carotovora subsp. carotovora from Iran. Biotechnology, 2005, 4(2): [8] Christensen P, Cook FD. Lysobacter, a new genus of non-fruiting, gliding bacteria with s high base ratio. International Journal of Systematic Bacteriology, 1978, 28(2): [9] Lee JW, Im WT, Kim MK, et al. Lysobacter koreensis sp. nov. isolated from a ginseng field. Int J Syst Evol Microbiol, 2006, 56(1): [10] Jochum CC, Osborne LE, Yuen GY. Fusarium head blight biological control with Lysobacter enzymogenes C3. Biological Control, 2006, 39(3): [11] Md TI, Yasuyuki H. Possible role of xanthobaccins produced by Stenotrophomonas sp. strain SB-K88 in suppression of sugar beet damping-off disease. Applied and Environmental Microbiology, 1999, 65(10): [12] Giesler LJ, Yuen FY. Evaluation of Stenotrophomonas maltophilia strain C3 for biocontrol of brown patch disease. Crop Protection, 1998, 17(6): [13] Yuen GY, Steadman JR, Lindgren DT, et al. Bean rust biological control using bacterial agents. Crop Protection, 2001, 20(5): [14] Islam MT, Hashidoko Y, Deora A, et al. Suppression of damping-off disease in host plats by rhizoplane bacterium Lysobacter sp. strain SB-K88 in linked to plant colozation and antibiosis against soilborne peronosporomycetes. Applied and Environmental Microbiology, 2005, 71(7): [15] Folman LB, De Klein MJEM, Postma J, et al. Production

7 980 微生物学通报 2009, Vol.36, No.7 of antifungal compounda by Lysobacter enzymogenes isolate 3.1 T8 under different conditions in relation to its efficacy as a biocontrol agent of Pythium aphanidermatem in cucumber. Biological Control, 2004, 31(2): [16] JI GH, Wei LF, He YQ, et al. Biocontol of rice bacterial blight by Lysobacter antibioticus strain Biological control, 2008, 45(3): [17] Murray RGE, Doetsch RN, Robinow CF. Determinative and cytological light microscopy. In: Methods for General and Molecular Bacteriology. ed. Gerhardt P, Murray RG, Wood WA, et al. Washington DC: American society of Microbiology, pp [18],.. :, 2001, pp [19] Holt JG, Krieg NR, Pha S, et al. Bergey s Manual of Determinative Bacteriology. 9 th ed. William and Wilkins, Baltimore, USA, 1994, p.151. [20] Weisburg WG, Barns SM, Pelletier DA, et al. 16S ribosomal DNA amplification for phylogenetic study. Journal of Bacteriology, 1991, 173(2): 稿件书写规范高校教改纵横栏目简介及撰稿要求,,,,

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Microsoft Word - 廣效性植物源農藥之開發.doc 12 1 97 11 30 ( ) E-Mail wcho@mail.tajen.edu.tw 80 1000 26 (Trichoderma spp.) T20 T21 24 T21 T22 T26 46 63 33 19 600 100 800 800 1000 500 200 2000 Abstract In this study, chemical pesticide, antimicroorganisms