Identification Of An Antagonistic Bacillus Strain Against Sclerotinia Sclerotiorum And Study On The Antagonistic Compound

Sclerotinia sclerotiorum is a plant pathogenic fungus found in soil ubiquitously in many parts of the world. In China, this pathogen causes stem rot on oilseed rape (Brassica napus), and causes serious losses every year. Biological control is a useful approach compared to chemical fungicides against Scleriotinia diseases. In this paper, an antagonistic Bacillus strain X-01 which showed strong inhibition against S. sclerotiorum was screened out, and the taxonomy of X-01 strain was identified. The antagonistic compound produced by strain X-01 was isolated and identified. The potential for biological control of Scleriotinia disease on oilseed rape was evaluated. The main results were listed below.1. An antagonistic microorganism against S. sclerotiorum was obtained by dual-culture method, and was named X-01. It was identified to be Bacillus subtilis based on morphology observation, biochemical and physiological identification, phylogenetic analysis of 16S rDNA sequence.2. The antifungal activity of Bacillus subtilis X-01 was resistant to high temperature, a wide range of pH and the action of many hydrolytic enzymes. The antifungal compounds produced by Bacillus subtilis X-01 passed through an ultrafiltration cartridge with molecular weight cutoff (MWCO) of 100 kDa, and was retained by an ultrafiltration cartridge with MWCO of 30 kDa. But, when the culture filtrate was added with methanol at a final concentration of 50%, the antifungal activity was detected in the filtrates of an ultrafiltration cartridge with MWCO of 30 kDa. All the results above implied the antifungal compounds belong to lipopeptides.3. Utrafiltration, solid phase extraction and high performance liquid chromatography were combined to isolate the antifungal compound produced by Bacillus subtilis X-01. Matrix assisted laser desorption ionization-time of flight mass Spectrometry (MALDI-TOF MS) analysis showed the isolated active compound had molecular mass of 1042.9 Da. Electrospray ionization(ESI) coupled with collision activated dissociation(CAD) mass Spectrometry analysis was used to sequence the structure of purified compound. Typical b-and y-type fragments showed that the compound had a primary structure of Pro-Asn-Ser-βAA-Asn-Tyr-Asn-Gln (βAA representedβ-amino fatty acid with 14 carbon atoms), which was consistent with lipopeptide iturin A₂.4. The isolated compound produced by Bacillus subtilis X-01 at a concentration of 100μg/mL had an inhibitory effect to S. sclerotiorum on the leaves of oilseed rape with the suppression rate of 100% in vitro. The suppression rate of the compound to S. sclerotiorum remained 40.7% at a concentration of 10μg/mL.5. The pictures of scan electron microscope after the treatment of the antagonistic compound produced by Bacillus subtilis X-01 to S. sclerotiorum showed that the hypha appeared rupture, distortion, pore formed on the cell wall, protoplasm condensed and the leak of the contents in cells.