Unraveling the mode of antifungal action of Bacillus subtilis and Bacillus amyloliquefaciens as potential biocontrol agents against aflatoxigenic Aspergillus parasiticus

Some filamentous fungi are plant pathogens that cause serious economic losses in foodstuffs and agricultural commodities. In the present study, the antifungal effects of culture filtrates of soil strains of Bacillus subtilis and Bacillus amyloliquefaciens were studied against some common field-pathogenic fungi with special reference to the possible mode of actions against Aspergillus parasiticus NRRL2999. The fungal growth and aflatoxin production were determined by microbioassay technique. Changes in membrane ergosterol content, mitochondrial dehydrogenase activity, and chitin and β-1,3-glucan content of the fungal cell wall were evaluated as the different possible targets in the presence of various concentrations of bacterial culture filtrates. According to the results, both bacteria showed strong antifungal activity against the broad spectrum of filamentous fungi. They were able to suppress A. parasiticus growth (up to 92%) and aflatoxin production (up to 100%) and showed good proteolytic activities (up to 10.5 ± 0.4 mm clear zones on gelatin agar). Both antagonistic bacteria decreased ergosterol content of the fungal cell membrane of A. parasiticus (9.0-80%) in a dose-dependent manner. They also affected dehydrogenase activity of fungal cell mitochondria. Interestingly, by using 2500 μl of bacterial culture filtrate, the slight resume of fungal growth and cell wall compositions, referred as the paradoxical effect, was exhibited. Scanning and transmission electron microscopy showed drastic changes in structure and shapeof the treated fungal hyphae and vesicles including folding, wrinkling, cell depletion and vacuolization. Altogether, our results show B. subtilis and B. amyloliquefaciens target different sites in the fungal cells and thus, they could act as biological control agents to combat A. parasiticus growth and subsequent aflatoxin contamination of crops and agricultural commodities.