sfp基因转化增强了Bacillus subtilis 168的定殖能力和对黄瓜茎内枯萎病菌的抑制作用

doi:10.16409/j.cnki.2095-039x.2015.06.012

摘要/Abstract

摘要： 枯草芽胞杆菌B006是一株防治黄瓜枯萎病的高效菌株,可产生脂肽类抗生素表面活性素surfactin和芬枯草菌素fengycin。为深入了解surfactin在根际的作用,本研究通过同源重组的方法将生防芽胞杆菌B006菌株中编码4'-磷酸泛酰巯基乙胺转移酶Sfp的sfp基因整合到不产生surfactin的模式菌株B.subtilis168(B168)基因组中,获得了重组菌株B168S。重组菌株B168S在1%淀粉平板上不水解淀粉,在5%血平板上产生透明溶血圈;HPLC-ESI-MS检测表明菌株B168S在牛肉膏蛋白胨培养液中培养48 h后可产生surfactin。平板对峙试验表明菌株B168S在黄瓜根分泌物培养基上对Fusarium oxysporum f. sp.cucumerinum(Foc)菌丝生长有一定的抑制作用,抑制率R2/R1为1.22;平板菌落计数法测定表明,将菌悬液(10⁶ cfu/mL)浸泡90 min的黄瓜种子播种于无菌石英砂3 d后,菌株B168S在黄瓜根基部和根尖的定殖量分别为菌株B168的2~3倍和9~10倍;温室盆栽试验表明,黄瓜苗用浓度为106 cfu/mL的B168S和B168菌悬液蘸根后,移栽到接种Foc孢子(10⁵孢子/g)的病土中,移栽后第3周菌株B168S和B168处理的防效分别为21.1%和17.9%;对不同高度黄瓜茎中Foc的组织分离结果表明,菌株B168S处理后Foc在黄瓜茎内蔓延的相对高度低于菌株B168和Foc处理。上述结果进一步明确了surfactin可明显促进芽胞杆菌在黄瓜根部的定殖,并通过抑制枯萎病菌的侵染和在黄瓜茎中的蔓延增强对黄瓜枯萎病的防治效果。

关键词: 同源重组, sfp 基因, 芽胞杆菌, surfactin, 黄瓜枯萎病菌

Abstract: B. subtilis B006 is an effective biocontrol bacterium of fusarium wilt diseases by producing surfactin and fengycin. To elucidate the function of surfactin in vivo, the complete sfp gene encoding protein of PPantransferase Sfp to activate the surfactin synthetase, was amplified from B. subtilis B006. A transformant strain B168S was constructed through inserting the sfp gene into the B. subtilis strain 168 by using homologous recombination method. The recombinant B168S did not hydrolyze starch on 1% starch medium, but producing hemolysis circle around its colonies on 5% blood plates. Detection by HPLC-ESI-MS showed that B168S produced surfactin after 48 h growth in nutrient broth, while B168 did not. Strain B168S suppressed the mycelial growth of Fusarium oxysporum f. sp. cucumerinum (Foc) on cucumber root exudate (CRE) medium plates with inhibitory rate 1.22. Colonization tests by dilution plating method indicated that the population of B168S colonized on cucumber root bases and root tips increased by 2-3 times and 9-10 times in comparison with B168 at 3rd day after germination when cucumber seeds soaked with 106 cfu/mL bacterial suspensions for 90 mins were sown in quartz sand. Greenhouse experiments showed that control efficacy of B168S and B168 against fusarium wilt disease was 21.1% and 17.9%, respectively, at the third week after transplantation of cucumber seedlings immersed with 10⁶ cfu/mL bacterial suspensions and transplanted to the substrate inoculated with Foc at 10⁵ spores/g of soil. The isolation of Foc from cucumber stems at different height showed that B168S was able to suppress the infection and the spread of Foc in cucumber, compared with the control and B168 treatments. In a conclusion, surfactin promoted B. subtilis colonization on cucumber roots and played an important role in suppressing the infection and spread of Foc in cucumber.

Key words: homologous recombination, sfp gene, Bacillus subtilis, surfactin, Fusarium oxysporum f. sp. Cucumerinum

中图分类号:

S432
S476.8

高毓晗, 李世东, 郭荣君. sfp基因转化增强了Bacillus subtilis 168的定殖能力和对黄瓜茎内枯萎病菌的抑制作用[J]. 中国生物防治学报, 2016, 32(1): 76-85.

GAO Yuhan, LI Shidong, GUO Rongjun. Transformation of sfp Gene into Bacillus subtilis 168 Promotes Its Colonization on Cucumber Roots and Suppression of Fusarium oxysporum f. sp. cucumerinum in Cucumber Stems[J]. journal1, 2016, 32(1): 76-85.

参考文献

[1] Nagórska K, Bikowski M, Obuchowski M. Multicellular behaviour and production of a wide variety of toxic substances support usage of Bacillus subtilis as a powerful biocontrol agent[J]. Acta Biochimica Polonica, 2007, 54:495-508.
[2] Szczech M, Shoda M. The effect of mode of application of Bacillus subtilis RB14-C on its efficacy as a biocontrol agent against Rhizoctonia solani[J]. Journal of Phytopathology, 2006, 154:370-377.
[3] Nihorimbere V, Cawoy H, Seyer A, et al. Impact of rhizosphere factors on cyclic lipopeptide signature from the plant beneficial strain Bacillus amyloliquefaciens S499[J]. FEMS Microbiology Ecology, 2012, 79:176-191.
[4] Kinsella K, Schulthess C P, Morris T F, et al. Rapid quantification of Bacillus subtilis antibiotics in the rhizosphere[J]. Soil Biology and Biochemistry, 2009, 41(2):374-379.
[5] 杨琦瑶, 索雅丽, 郭荣君, 等. 枯草芽孢杆菌B006对黄瓜枯萎病菌和辣椒疫霉病菌的抑制作用及其抗菌组分分析[J]. 中国生物防治学报, 2012, 28:235-242.
[6] Ongena M, Jourdan E, Adam A, et al. Surfactin and fengycin lipopeptides of Bacillus subtilis as elicitors of induced systemic resistance in plants[J]. Environmental Microbiology, 2007, 9:1084-1090.
[7] Zeriouh H, de Vicente A, Perez-Garcia A, et al. Surfactin triggers biofilm formation of Bacillus subtilis in melon phylloplane and contributes to the biocontrol activity[J]. Environmental Microbiology, 2014, 16:2196-2211.
[8] Chen Y, Yan F, Chai Y, et al. Biocontrol of tomato wilt disease by Bacillus subtilis isolates from natural environments depends on conserved genes mediating biofilm formation[J]. Environmental Microbiology, 2013, 15:848-864.
[9] Gao S, Wu H, Wang W, et al. Efficient colonization and harpins mediated enhancement in growth and biocontrol of wilt disease in tomato by Bacillus subtilis[J]. Letters in Applied Microbiology, 2013, 57:526-533.
[10] Ghelardi E, Salvetti S, Ceragioli M, et al. Contribution of surfactin and SwrA to flagellin expression, swimming, and surface motility in Bacillus subtilis[J]. Applied and Environmental Microbiology, 2012, 78:6540-6544.
[11] Cawoy H, Mariutto M, Henry G, et al. Plant defense stimulation by natural isolates of Bacillus depends on efficient surfactin production[J]. Molecular Plant-Microbe Interactions, 2014, 27:87-100.
[12] Li D Q, Nie F Y, Wei L H, et al. Screening of high-yielding biocontrol bacterium Bs-916 mutant by ion implantation[J]. Applied Microbiology and Biotechnology, 2007, 75:1401-1408.
[13] 贾珂, 李世东, 刘桂君, 等. 枯草芽孢杆菌B006产surfactin突变株特性及其对黄瓜枯萎病的抑制能力[J]. 中国生物防治学报, 2013, 29(4):538-546.
[14] Kunst F, Ogasawara N, Moszer I, et al. The complete genome sequence of the gram-positive bacterium Bacillus subtilis[J]. Nature, 1997, 390(6657):249-256.
[15] Nakano M M, Corbell N, Besson J, et al. Isolation and characterization of sfp:a gene that functions in the production of the lipopeptide biosurfactant, surfactin, in Bacillus subtilis[J]. Molecular and General Genetics, 1992, 232:313-321.
[16] Lambalot R H, Gehring A M, Flugel R S, et al. A new enzyme superfamily-the phosphopantetheinyl transferases[J]. Chemistry and Biology, 1996, 3:923-936.
[17] Mootz H D, Finking R, Marahiel M A. 4'-phosphopantetheine transfer in primary and secondary metabolism of Bacillus subtilis[J]. The Journal of Biological Chemistry, 2001, 276:37289-37298.
[18] 刘丽霞. Surfactin合成酶相关基因研究[D]. 南京:南京农业大学, 2012.
[19] 魏艳, 张丹, 蔡恒, 等. 适用于野生型枯草芽孢杆菌转化有机溶剂方法的建立和优化[J]. 生物学通报, 2011, 12:42-45.
[20] Ongena M, Jacques P. Bacillus lipopeptides:versatile weapons for plant disease biocontrol[J]. Trends in Microbiology, 2008, 16:115-120.
[21] Kamilova F, Kravchenko L V, Shaposhnikov A I, et al. Organic acids, sugars, and L-tryptophane in exudates of vegetables growing on stonewool and their effects on activities of rhizosphere bacteria[J]. Molecular Plant-Microbe Interactions, 2006, 19:250-256.
[22] Coutte F, Leclere V, Bechet M, et al. Effect of pps disruption and constitutive expression of srfA on surfactin productivity, spreading and antagonistic properties of Bacillus subtilis168 derivatives[J]. Journal of Applied Microbiology, 2010, 109:480-491.
[23] Julkowska D, Obuchowski M, Holland I B, et al. Branched swarming patterns on a synthetic medium formed by wild-type Bacillus subtilis strain 3610:detection of different cellular morphologies and constellations of cells as the complex architecture develops[J]. Microbiology, 2004, 150:1839-1849.
[24] 李世贵. 防治黄瓜枯萎、青椒疫病木霉菌的研究[D]. 北京:中国农业科学院, 2005.
[25] Burgess L W, Knight T E, Tesorieo L, et al. Diagnostic Manual for Plant Diseases in Vietnam[M]. Canberra:Australian Centre for International Agricultural Research, 2008, 92-94.
[26] Chen H, Wang L, Su C X, et al. Isolation and characterization of lipopeptide antibiotics produced by Bacillus subtilis[J]. Letters in Applied Microbiology, 2008, 47:180-186.
[27] Kloepper J W, Ryu C M, Zhang S. Induced systemic resistance and promotion of plant growth by Bacillus spp.[J]. Phytopathology, 2004, 94:1259-1266.
[28] 董伟欣, 李宝庆, 李社增, 等. 脂肽类抗生素fengycin在枯草芽胞杆菌NCD-2菌株抑制番茄灰霉病菌中的功能分析[J]. 植物病理学报, 2013, 43(4):401-410.