Purification and Structural Analysis of Surfactin Produced by Endophytic Bacillus subtilis EBS05 and its Antagonistic Activity Against Rhizoctonia cerealis

College of Plant Protection, Henan Agricultural University, Zhengzhou, Henan 450002, China(Received on March 9, 2011; Revised on July 22, 2011; Accepted on August 19, 2011)Bacillus subtilis EBS05, an endophytic bacteria strainisolated from a medicinal plant Cinnamomum camphor,can produce antagonistic compounds that effectivelyinhibit plant pathogenic fungi. The greenhouse experi-ments showed that wheat sharp eyespot disease (WSED)was reduced by 91.2%, 88.2% and 43.0% after thetreatment with fermentation broth, bacteria-free filterand a fungicide fludioxonil, respectively. The culturebroth of strain EBS05 can more effectively controlWSED than can fludioxonil. The fermentation brothand bacteria-free filter ability to suppress WSED wasnot significantly different, suggesting that an activesecreted substance played a major role in controllingWSED. Separation and purification of the active com-pounds was carried out by serial processes, includinghydrochloric acid (pH 2.0) treatment, methanol ex-traction and Sephadex LH-20 column chromatography,silica gel column chromatography and reverse-phasehigh-pressure liquid chromatography (HPLC), respec-tively. The purified compounds, one of active peaks inthe HPLC spectrum, were obtained from the collection.Analysis of the chemical structures by time-of-flightmass spectrometry (TOF-MS) and electrospray ioni-zation mass spectrometry/mass spectrometry (ESI-MS/MS) showed that the active substances produced by theendophytic bacteria EBS05 are mixture of the β-hydr-oxy-C12~C15-Leu