Inhibition of antifungal substances from Bacillus amyloquefaciens B15 against Botrytis Cinerea—the agent of “gray mold” of grape

Abstract:

[Objective] The present study aimed to investigate the inhibition of cyclic lipopeptides (iturin A and fengycin) produced by Bacillus amyloquefaciens B15 against Botrytis cinerea by using laser confocal scanning microscopy and fluorescence microscopy. [Methods] The agar diffusion method was applied to investigate the antifungal activity of B15 strain against Botrytis cinerea. A 7-mm agar plug of Botrytis cinerea was placed on the PDA plate containing B15 culture supernatant at the concentration of 1:20. The hyphal viability was determined by staining the hyphae of Botrytis cinerea with trypan blue. Then the classical cell apoptotic assay was applied by staining cell nucleus, reactive oxygen species, calcium ions, and phosphatidylserine of hyphae with DAPI, Dihydrorhodamine 123, fluo-3/am and Annexin V-PI probe. [Results] The antagonistic assay showed that the B15 strain displayed evident antifungal activity against Botrytis cinerea. From the trypan blue assay, it was evident to observe the distorted hyphal structure of Botrytis cinerea in treated group, including conglobation of branch tips, swollen hyphae, hyphal vacuolation, and shrinkage under fluorescence microscopy. The laser confocal scanning microscopy observation of Botrytis cinerea showed that the typical cell apoptosis pattern appeared in the treated group, including the nuclear fragmentation and condensation, accumulation in reactive oxygen species and Ca²⁺, and the flip-flop of phosphatidylserine. [Conclusion] The combined results of the trypan blue assay and cell apoptotic assay indicated that B15 culture supernatant inhibited the growth of Botrytis cinerea in the way of inducing apoptotic-like cell death in Botrytis cinerea, instead of damaging the cell membrane and directly contributing to the death of mycelium.

Key words:Bacillus amyloquefaciens B15;apoptotic-like programmed cell death;mechanisms of inhibition;laser confocal scanning microscopy;Botrytis cinerea