Construction Of Surfactin Producing Strain Of Bacillus Subtilis 168 And Its Control Effect On Cucumber Fusarium Wilt

Fusarium wilt of cucumber caused by Fusarium oxysporum f. sp. cucumerinum(Foc) resulted in serious soil borne disease. Bacillus subtilis B006 is an efficient strain to suppress cucumber fusarium wilt disease. It can produce lipopeptide antibiotics surfactin and fengycin. Previous researches have proved that surfactin can promote the biofilm formation and colonization of Bacillus on plant roots, and induce plant to produce systemic resistance. But no evidence proved that surfactin can inhibit the infection of the rhizosphere pathogens, and suppress the spread of pathogens within plant stems to reduce the occurrence and delay the disease development.To elucidate the function of surfactin in vivo, the complete sfp gene, important and necessary for surfactin synthesis, was first amplified from B. subtilis B006 which encodes protein of PPantransferase Sfp to activate the surfactin synthetase. Then the sfp gene was ligated with pDG1730, and a transformant strain B168 S was constructed through inserting the sfp gene into the B. subtilis strain 168 by using homologous recombination method. Recombinant strain B168 S was selected by showing negative reaction to hydrolyze starch on 1% starch medium, but positive reaction of producing hemolysis circle around the colonies on 5% blood plates. Further detection by HPLC-ESI-MS showed that B168 S produced surfactin after 48 h growth in nutrient broth, compared with B168 strain which cannot produce surfactin because of the frame shift mutation of sfp gene.Determination of biological characteristics of recombinant strain B168 S and wild strain B168 showed that the colony morphology of strain B168 S was different with B168 on cucumber root exudates(CRE), indicating by the thicker colony of B168 S. And the biofilm of B168 S was thicker than B168 after static culturing in LB broth for 24 h at 37℃. Strain B168 S also exhibited inhibitory activity to suppress the mycelial growth of Foc on CRE plates with the inhibitory rate(R2/R1) of 1.22., while strain B168 didn’t show any inhibitory activity.Determination of distribution of starins B168 S and B168 colonized on different part of cucumber roots by tissue printing method on CRE plates indicated that large amount of B168 S colonized on the middle and the tip of the roots in comparison with strain B168. The population of strain B168 S colonized on cucumber roots was further determined by using dilution plating method. The cucumber seeds were sown in quartz sand after soaking with 106 cfu·mL-1 bacterial suspensions for 90 mins. Three days after germination, the population of B168 S colonized on cucumber root bases and root tips increased by 2~3 times and 9~10 times compared with strain B168.The efficacy of strain B168 S to control fusarium wilt disease was further tested in the greenhouse. Cucumber seedlings were immersed with 106 cfu·mL-1 bacterial suspensions of B168 S and B168, and were transplanted to the substrate inoculated with Foc spores at the concentration of 105 spores·g-1. At the third week after transplantation, the control efficacy of B168 S and B168 was 21.1% and 17.9%, respectively. The isolation of Foc from cucumber tissue at different height showed that B168 S was able to suppress the infection and the spread of Foc in cucumber, compared with the control and B168 treatments. In a conclusion, surfactin can promote B. subtilis colonization on cucumber roots and plays an important role in suppressing the infection and spread of Foc in cucumber.Root exudates recomposed medium with citric acid, malic acid, succinic acid as unitary organic acid were used to culture Bacillus subtilis B006. The results of HPLC detection of surfactin produced by B006 showed that three kinds of organic acid could improve the surfactin production and change the proportion of the homologues of surfactin. Addition of surfactin crude extract promoted the formation of Bacillus biofilms. And surfactin A and B extracted from glucose or succinate root exudates(RE)broth had different effect on the promotion of B168 and B168 S biofilm formation. The surfactin extract A and B had a significant promotion to the biofilm formation over 50 and 10 μM, respectively.The results in this study further clarified that surfactin can obviously promote the Bacillus colonization in cucumber root and suppress the infection and delay the spread of Foc in cucumber stem to enhance the inhibitory effect of cucumber on fusarium wilt. These results will benefit our understanding of the surfactin function in the rhizosphere, and providing useful information for the application of Bacillus.