Growth inhibitory properties of Bacillus subtilis strains and their metabolites against the green mold pathogen (Penicillium digitatum Sacc.) of citrus fruit

Twenty three strains of Bacillus spp. screened from 205 Bacillus spp. isolated from soil, showed antagonistic activities in vitro towards the Penicillium digitatum pathogen, a cause of citrus fruit rot disease. Culture supernatants from nine strains caused >80% inhibition of P. digitatum growth when they were serially diluted to 1:32. Volatile compounds produced by these strains also caused 30–70% inhibition of fungal growth. An ethanol extract from a Bacillus subtilis 155 cell-free supernatant referred to as secondary metabolites (SMs) produced the best inhibitory effect on mycelial growth and spore germination of the fungus with EC50 values of 77.26 and 82.10 μgm L−1, respectively. Inhibitory compounds, separated from the SMs by preparative thin-layer chromatography (CHCl3/MeOH/H2O: 65/25/4, v/v/v), had Rf values of 0.14, 0.28, 0.31, 0.49, and 0.64 with EC50 values of 95.73, 14.07, 15.19, 108.59, and 99.98 μg mL−1, respectively. Protein precipitated with 80% saturated ammonium sulphate, from the culture supernatant, had an EC50 of 288 μg mL−1. After native polyacrylamide gel electrophoresis of this protein the antifungal protein activity was detected only in the lowest band. Inoculation of a suspension of P. digitatum conidia (104 conidia mL−1) onto wounded citrus fruit induced disease symptoms at day 3 and decay at day 5. Inoculation with 20 μL of a 108 CFU mL−1B. subtilis endospore suspension 24 h prior to fungal spore inoculation decreased disease incidences by 86.7%, and disease symptoms were delayed by 6 days and decay symptoms to day 9. Addition of the SMs (10 mg mL−1), simultaneously with the fungus decreased disease incidence by 72.5%, delayed disease symptoms up to 5 days after inoculation, and no sign of decay was observed up to 9 days. The average lesion diameters observed from treatments with bacterial endospores, SMs and a control fungicide, imazalil were significantly different from the size of the wounds in the control set treated only with fungal conidia. B. subtilis 155 and its antibiotics are considered to be potent biological control agents to suppress growth of P. digitatum in the postharvest protection of citrus.