Scientia Agricultura Sinica ›› 2013, Vol. 46 ›› Issue (18): 3776-3783.doi: 10.3864/j.issn.0578-1752.2013.18.005

• PLANT PROTECTION • Previous Articles     Next Articles

Plasmid Elimination of Bacillus subtilis NJ-18 and Its Colonization on Wheat Roots

 LU  Jing-Le, YU  Xin-Yan, HOU  Yi-Ping, WANG  Jian-Xin, ZHOU  Ming-Guo, CHEN  Chang-Jun   

  1. College of Plant Protection, Nanjing Agricultural University/Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing 210095
  • Received:2013-03-01 Online:2013-09-15 Published:2013-05-22

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Abstract: 【Objective】Bacillus subtilis strain NJ-18, isolated from the soil of oilseed rape field, has a broad spectrum antibacterial activity and a great potential for biocontrol. In order to study the interaction between NJ-18 and plants, NJ-18 was marked with a green fluorescent protein (gfp) gene and its colonization ability on wheat roots was observed with a laser scanning confocal microscope. 【Method】 The plasmids of NJ-18 were cured by sodium dodecyl sulfate (SDS) and gfp was transformed into the strain with a chemical method. Colonization ability of NJ-18 on wheat roots was observed with a laser scanning confocal microscope. 【Result】 By a chemical method, gfp was sucessfully transformed into wild strain NJ-18 and C136, a plasmid cured strain from NJ-18. The transformation efficiency of C136 could be up to 3.42×105 cfu/μg of plasmid DNA and was about 10-fold of that of NJ-18. Compared with NJ-18, C136 had the same antibacterial activity, but its growth was inhibited in a certain extent. NJ-18 could attach on the pathogen microbial cells, resulting in mycelium enlargement and malformations. NJ-18 was able to colonize in the phloem and xylem of wheat roots on 7 days after inoculation. 【Conclusion】 The chemical transformation method is suitalbe for strain NJ-18. NJ-18 can colonize on wheat roots.

Key words: NJ-18 , plasmid-cured , chemical transformation , GFP , colonization

[1]Cavaglieri L, Orlando J, Rodriguez M I, Chulze S, Etcheverry M. Biocontrol of Bacillus subtilis against Fusarium verticillioides in vitro and at the maize root level. Research in Microbiology, 2005, 156(5/6): 748-754.

[2]Estevez-de-Jensen C, Percich J A, Graham P H. Integrated management strategies of bean root rot with Bacillus subtilis and Rhizobium in Minnesota. Field Crops Research, 2002, 74(2/3): 107-115.

[3]Sharma N, Sharma S. Control of foliar diseases of mustard by Bacillus from reclaimed soil. Microbiological Research, 2008, 163(4): 408-413.

[4]Scherm H, Ngugi H K, Savelle A T, Edwards J R. Biological control of infection of blueberry flowers caused by Monilinia vaccinii- corymbosi. Biological Control, 2004, 29(2): 199-206.

[5]Ahimou F, Jacques P, Deleu M. Surfactin and iturin A effects on Bacillus subtilis surface hydrophobicity. Enzyme and Microbial Technology, 2000, 27(10): 749-754.

[6]PalBais H, Fall R, Vivanco J M. Biocontrol of Bacillus subtilis against infection of arabidopsis roots by Pseudomonas syringae is facilitated by biofilm formation and surfactin production. Plant Physiology, 2004, 134(1): 307-319.

[7]Raaijmakers J M, Vlami M, de Souza J T. Antibiotic production by bacterial biocontrol agents. Antonie van Leeuwenhoek, 2002, 81(1/4): 537-547.

[8]Liu Y F, Chen Z Y, Ng T B, Zhang J, Zhou M G, Song F P, Lu F, Liu Y Z. Bacisubin, an antifungal protein with ribonuclease and hemagglutinating activities from Bacillus subtilis strain B-916. Peptides, 2007, 28(3): 553-559.

[9]Aune T E V, Aachmann F L. Methodologies to increase the transformation efficiencies and the range of bacteria that can be transformed. Applied Microbiology and Biotechnology, 2010, 85(5): 1301-1313.

[10]Xue G P, Johnson J S, Dalrymple B P. High osmolarity improves the electro-transformation efficiency of the gram-positive bacteria Bacillus subtilis and Bacillus licheniformis. Journal of Microbiological Methods, 1999, 34(3): 183-191.

[11]Zhang G Q, Bao P, Zhang Y, Deng A H, Chen N, Wen T Y. Enhancing electro-transformation competency of recalcitrant Bacillus amyloliquefaciens by combining cell-wall weakening and cell-membrane fluidity disturbing. Analytical Biochemistry, 2011, 409(1): 130-137.

[12]Shriram V, Jahagirdar S, Latha C, Kumar V, Puranik V, Rojatkar S, Dhakephalkar P K, Shitole M G. A potential plasmid-curing agent, 8-epidiosbulbin E acetate, from Dioscorea bulbifera L. against multidrug-resistant bacteria. International Journal of Antimicrobial Agents, 2008, 32(5): 405-410.

[13]Mesas J M, Rodríguez M C, Alegre M T. Plasmid curing of Oenococcus oeni. Plasmid, 2004, 51: 37-40.

[14]Errampali D, Leung K, Cassidy M B, Kostrzynska M, Blears M, Lee H, Trevors J T. Application of the green fluorescent protein as a molecular marker in environmental microorganisms. Journal of Microbiological Methods, 1999, 35(3): 187-199.

[15]Bloemberg G V, O’Toole G A, Lugtenberg B J, Kolter R. Green fluorescent protein as a marker for Pseudomonas spp.. Applied and Environmental Microbiology, 1997, 63(11): 4543-4551.

[16]Liu X, Zhao H, Chen S. Colonization of maize and rice plants by strain Bacillus megaterium C4. Current Microbiology, 2006, 52(3): 186-190.

[17]Timmusk S, Grantcharova N, Wagner E G. Paenibacillus polymyxa invades plant roots and forms biofilms. Applied and Environmental Microbiology, 2005, 71(11): 7292-7300.

[18]龙海英, 刘衡川, 方梅. 质粒消除方法及消除效果的评价研究. 中国卫生检验杂志, 2007, 17(3): 413-415.

Long H Y, Liu H C, Fang M. Study of plasmid curing methods and appraisal of plasmid elimination effect. Chinese Journal of Health Laboratory Technology, 2007, 17(3): 413-415. (in Chinese)

[19]娄恺, 班睿, 赵学明. 细菌质粒的消除. 微生物学通报, 2002, 29(5): 99-103.

Lou K, Ban R, Zhao X M. Plasmid curing in bacteria. Microbiology, 2002, 29(5): 99-103. (in Chinese)

[20]章四平, 刘圣明, 王建新, 周明国. 枯草芽孢杆菌NJ-18发酵条件优化研究. 南京农业大学学报, 2010, 33(2): 58-62.

Zhang S P, Liu S M, Wang J X, Zhou M G. Optimizing fermentation condition for the antagonistic Bacillus subtilis NJ-18 strain. Journal of Nanjing Agricultural University, 2010, 33(2): 58-62. (in Chinese)

[21]Yang D J, Wang B, Wang J X, Chen Y, Zhou M G. Activity and efficacy of Bacillus subtilis strain NJ-18 against rice sheath blight and Sclerotinia stem rot of rape. Biological Control, 2009, 51(1): 61-65.

[22]Spizizen J. Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate. Proceedings of the National Academy of Sciences of the United States of America, 1958, 44(10): 1072-1078.

[23]杨冬静, 王建新, 周明国. Bacillus subtilis NJ-18菌株对番茄早疫病菌的颉颃作用研究. 微生物学通报, 2009, 36(8): 1166-1169.

Yang D J, Wang J X, Zhou M G. Biological Control of Alternaria solani by Bacillus subtilis NJ-18. Microbiology, 2009, 36(8): 1166-1169. (in Chinese)
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