Objective: The effects of repeated freeze-thaw cycles(F-Tc) on cell shape of fermented Bacillus subtilis and the inhibitory activity to Alternaria alternata, the primary infection pathogen of jujube fruit shrunken disease, were determined using an endophytic B. subtilis strain St-zn-34 isolated from winter jujube.
Methods: The fermented broth was freeze-thawed after batch fermentation of the test strain. The population of living bacteria and bacterial endospores were determined by dilution methods of plate counting, and the inhibitory activity to A. alternata was tested by filter paper disks with filtrate of fermented broth. The shape of B. subtilis with different freeze-thaw cycles was observed under environment scanning electron microscope.
Results: Changes of pH, living bacteria, bacterial endospore counts in the fermentation broth and the inhibitory activity of filtrate at different time generally increased first and decreased afterwards. The inhibitory activity at 60 h was higher than other time points. The bacterial cells fermented for 60 h were treated with F-Tc, and the living bacteria count and inhibitory activity decreased gradually when cells of B. subtilis were freeze-thawed for 3 cycles, but it had no significant difference (P>0.05) after being freeze-thawed for more than 3 times. With the increase of freeze-thawing times, the bacteria cells became smaller than the normal cells, the surface twisted with one or more depressions, and the jelly flowed out under environment scanning electron microscope. The inhibitory activity of filtrate was determined and it showed a broad-spectrum inhibitive capability against twelve species of plant pathogens. The treatment of different temperature and proteinase to the filtrate showed that the temperature below 60℃ did not affect the inhibitive activity and had no significant difference compared with that of the control. However, the inhibition decreased with increasing of the temperature above 80℃ and was significantly lower than that of control. The inhibitory activity of filtrate to A. alternata decreased by treatment with proteinase K.
Conclusion: Repeated freeze-thaw cycles could affect the shape of B. subtilis and reduced the inhibitory activity of filtrate to A. alternata.
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