Purification and characterization of a potential antifungal protein from Bacillus subtilis E1R-J against Valsa mali
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In order to identify the antagonistic substances produced by Bacillus subtilis E1R-J as candidate of biocontrol agents for controlling Apple Valsa Canker, hydrochloric acid precipitation, reverse phase chromatography, gel filtration, and ion exchange chromatography were used. The purified fraction EP-2 showed a single band in native-polyacrylamide gel electrophoresis (native-PAGE) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Fraction EP-2 was eluted from native-PAGE and showed a clear inhibition zone against V. mali 03-8. These results prove that EP-2 is one of the most important antifungal substances produced by B. subtilis E1R-J in fermentation broth. SDS-PAGE and Nano-LC–ESI–MS/MS analysis results demonstrated that EP-2 was likely an antifungal peptide (trA0A086WXP9), with a relative molecular mass of 12.44 kDa and isoelectric point of 9.94. The examination of antagonistic mechanism under SEM and TEM showed that EP-2 appeared to inhibit Valsa mali 03-8 by causing hyphal swelling, distortion, abnormality and protoplasts extravasation. Inhibition spectrum results showed that antifungal protein EP-2 had significantly inhibition on sixteen kinds of plant pathogenic fungi. The stability test results showed that protein EP-2 was stable with antifungal activity at temperatures as high as 100 °C for 30 min and in pH values ranging from 1.0 to 8.0, or incubated with each 5 mM Cu2+, Zn2+, Mg2+, or K+. However, the antifungal activity was negatively affected by Proteinase K treatment.
KeywordsEndophytic Bacillus subtilis Apple Valsa Canker (AVC) Biocontrol agents (BCAs) Antifungal substances
This study was supported by the Special Fund for Agro-Scientific Research in the Public Interest (No. 201203034), Science and Technology Innovation Research of Shaanxi (No. 2011KTZB02-02-02) and the National Natural Science Foundation of China (31101476). We are grateful to Dr. Ralf T. Voegele at Universität Hohenheim, Dr. Bing Liu (Jiangxi Agricultural University) and Prof. Heinrich Buchenauer (University Hohenheim) for comments and improvement of the manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare no conflict of interest.
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