| Bacillus subtilis NCD-2 was isolated from cotton rhizosphere and it showed strong inhibition against Verticillium dahliae, and significant control effect on cotton verticillium wilt.Characteristics of the crude antifungal protein produced by strain NCD-2 was evaluated under different culture conditions. The result showed that the strain could produce more antifungal substances under the optimum condition (cultured in NB broth for 2 d with180rpm under 30℃and pH 7.0).Ammonium sulfate precipitates of strain NCD-2 was collected and it showed strong inhibition effect against V. dahliae. The antifungal activity of the substrate treated with RNase was no significant difference compared with that of control. Meanwhile, the antifungal activities of the substance treated with Proteinase K and Trypsin were significantly decreased, respectively. And the antifungal activity of the substrate treated with 60℃was no significant difference, while the antifungal activity of the substrate treated with 80℃and 100℃were significantly different. However, the crude protein extract still had antifungal activity even when it was treated at 120℃for 30 minutes. The results indicated that the crude extract of strain NCD-2 included several different antifungal substances.The antifungal protein precipitated by salt out with 30%～80% ammonium sulfate was purified by chromatography in this paper, three parts were obtained from DEAE-Sepharose Fast the Flow, and the second part showed antifungal activity. After purified through Sephacry S-100, the first and the third part showed antagonistic effect against V. dahliae and the two parts had achieved electrophoresis purify after analyzed by RPC.In this paper, lipopeptide antibiotics crude extracted by hydrochloric acid was composed of many components after analyzed by RPC. One of them had the similar retention time to Iturin, and ESI-MS analysis showed that the antifungal substance contained three homologues of Iturin.
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