Effect Of Surfactin Excreted By Bacillus Subtilis B006on The Control Of Cucumber Fusarium Wilt

Bacillus subtilis is widely used as a biocontrol agent for plant disease control. It can produce24antibiotics, of them, surfactin is a cyclopeptide with surface activity and bactericidal effect. In order tounderstand the importance of surfactin excreted by B. subtilis in its colonization and interaction withFusarium oxysporum f. sp. cucumerinum （FOC） in natural soil, B.subtilis B006was implanted bynitrogen ions, and three genetically stable surfactin mutants, B841, B73and B1020, were selected. Theireffects on the population and control efficacy of FOC were studied herein.HPLC-ESI-MS analysis of the24h culture in NB medium showed that in comparison with thewild strain B006, the amounts of surfactin excreted by mutants B841and B73increased by15.9%and14.8%, respectively, while that of B1020decreased by85.2%. The amounts of fengycin produced by thethree mutants were similar to that of strain B006. Mass spectrum analysis indicated that the constituentsof surfactin excreted by the mutants changed. The surfactin of B006had a mass spectrum with m/z at995,1009,1023,1037and1051, while m/z at995was absent in B841surfactin and only m/z at1037and1051was detected in mutant B1020surfactin. Biological characterization indicated that the colonymorphology of all mutants changed, their spore-forming rates reduced, and the life cycle of B73andB1020changed.Using the optimized real-time PCR program, colonization of strain B006and mutants B841andB1020in cucumber rhizosphere was examined by soaking seeds in106cfu·g^-1bacterial suspension. At1week, the population of B006and B841increased by13.8%and22.7%, respectively, while that ofB1020decreased by2.3%. This result corresponds to the surfactin-excreting ability of strain B006andits mutants B841and B1020in nutrient broth. At5week, all strains were similar in populations andproduced similar amounts of surfactin in cucumber rhizosphere.Greenhouse test in substrate with addition of strain B006and its mutants B841and B1020suspension at106cfu·g^-1showed that their control efficacy varied with the development of Fusariumwilt disease in cucumber. B006suppressed the wilt disease at1week with the control efficacy of above70%and still reached45.4%at5week. B841and B1020had little effect on disease control. Real-timePCR analysis indicated that Bacillus addition in nursery substrate reduced the FOC population incucumber stem, and B006gave the best performance. In cucumber rhizosphere of each Bacillustreatment, the population of FOC and the disease seriousness were negatively correlated to the highamount of Bacillus. In treatments with same Bacillus strain, the population of FOC and the diseaseseriousness were negatively correlated to the amount of surfactin.Inoculation of FOC and Bacillus suspension in natural soil showed that strain B006effectivelysuppressed the cucumber wilt disease with the best control efficacy, while B841and B1020had nocontrol capacity. Real-time PCR analysis showed that the populations of Bacillus and FOC in differenttreatments were similar in cucumber rhizosphere1week after transplantation. At3week, the amount ofB006in rhizosphere increased to8.71×10⁴cfu·g^-1, while FOC population was the least, as low as3.39 ×10³ng·g^-1. At5week, the amount of B006reduced to5.37×10⁴cfu·g^-1, and the FOC populationraised to1.82×10⁵ng·g^-1. Real-time PCR analysis of FOC population in cucumber stem showed thatstrain B006suppressed the spread and multiplication of FOC in the plants. The population of FOC wasthe least at1and3weeks, and lower in the upper leaves with Bacillus than that of blank control at5week. The amount of surfactin in cucumber rhizosphere of all treatments had significant differenceamong B006, B841and B1020. This result indicated that the surfactin amount didn’t affect the controlefficacy directly.In a summary, this study reveals that the control efficacy of Bacillus was not directly related to itssurfactin-excreting ability in nutrient broth. However, in treatments with the same Bacillus strain, thepopulation of FOC and the disease seriousness were negatively correlated to the amount of surfactin.These results provide profound information for the better understanding on the suppressing mechanismof Bacillus in natural soil and the application technique of Bacillus agents in field.