The antifungal mechanism of Bacillus subtilis against Pestalotiopsis eugeniae and its development for commercial applications against wax apple infection

The inhibition zones of three Bacillus subtilis isolates, BS-99, BS-23857 and BS-33608 against Pestalotiopsis eugeniae were, 13.5, 0, and 0 mm, respectively. BS-99 showed the strongest inhibitory activity, whereas, no inhibitory activity was reported for BS-23857 or BS-33608. The spore germination rates of P. eugeniae were not significantly inhibited by n-hexane, ethyl acetate (EtOAc), or methanol (MeOH) extracts of the fermentation broths of BS-99, BS-23857, and BS-33608. However, the hyphae of P. eugeniae became swollen and malformed after 12 h and stopped growing after 30 h of treatment with the MeOH extract of the BS-99 fermentation broth. Analysis by polymerase chain reaction (PCR), blood agar test plates, and high performance liquid chromatography (HPLC) indicated that, BS-99 produced the antibiotics, iturin A and surfactin, whereas, BS-23857 and BS-33608 only produced surfactin. These data suggest that, the antifungal activity of the B. subtilis BS-99 isolate against P. eugeniae is activated only when both iturin A and surfactin are present and not by surfactin alone. Unveiling the antifungal mechanism of B. subtilis, BS-99 could promote its commercial development and application as a biofungicide for controlling P. eugeniae infections of the wax apple, which is a highly valued fruit in Taiwan.