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

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

Abstract

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

CLC Number:

S432
S476.8

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.

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