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 Cu(2+), Zn(2+), Mg(2+), or K(+). However, the antifungal activity was negatively affected by Proteinase K treatment.
Keywords: Antifungal substances; Apple Valsa Canker (AVC); Biocontrol agents (BCAs); Endophytic Bacillus subtilis.