Inhibitory activity of Bacillus sp. strains and their metabolites against Penicillium digitatum and P. italicum, causal agents of green and blue mold on lemons

GRAMAJO, FM; ZULUAGA-ACOSTA, J; ADLER, C; FARIZANO, JV; RAPISARDA, VA; VOLENTINI, SI; DEBES, MA; CERIONI, L

Congreso; LVIII Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research; 2022

Argentine citrus production must face significant economic losses caused by postharvest diseases, mainly green mold (P. digitatum) and blue mold (P. italicum). Synthetic fungicides are used to control these fungal pathogens but, their widespread use has led to appearance of resistant fungal isolates. Therefore, there is an urgent need to develop safe alternatives strategies to control citrus postharvest diseases. Biopreservation, the control of one organism by another, has received much attention in the last years. Bacillus sp. are a promising candidates to be used as fungal antagonists, since they have been reported to have antimicrobial properties. Thus, the aim of this study was to evaluate the potential antifungal activity of FZB42 (B. velezensis), the local isolate CA1 (B. velezensis) and the commercial strain of B. subtilis QST713 against P. digitatum (PD) and P. italicum (PI), and to determine the appropriate media culture to produce their antifungal metabolites. The inhibitory activities on the growth of PD and PI of the Bacillus sp strains were assayed by the dual culture technique method on APG medium. All strains exhibited a strong micelial growth inhibition of both fungi. To evaluated the media influence in the production of antifungal metabolites, FZB42, CA1 and B QST713 were grown in LB, M9 or PG medium at 30°C. After 12h, cells were removed by centrifugation, and cell-free supernatants (CFS) were obtained by filtration and separated in 1x or concentrated to 10X. The antifungal activity of each CFS was evaluated in a 96-well polystyrene microtiter plate containing the fungal conidial suspensions adjust to 105 UFC/ml and the different CFS 1X (50:50). Microplates were incubated during 5d at 22°C and the conidia germination evaluation were performed by observation using an inverted light microscope. Results showed that, after 1d of incubation, CFS-PG of all Bacillus sp. strains inhibited conidia germination of both fungi being CFS-PG of FZB42 the most active, also showing that the conidia structure was altered by deformation and vacuolization. In the CFS-LB and CFS-M9 of all strains the conidia germination was retard, since the growth was observed after 5d of incubation. Finally, to detect CFS- PG 10x (of all strains) potential application for the prevention of green mold, assays were performed on lemons. For this, each lemon was wounded with a needle, 10μL of the different PG-CFS 10X were inoculated into each hole and fruits were air-dried for 1h. Then, 10μL of PD conidia suspension adjusted to 105 UFC/ml were added to each wound. After 7 d of incubation, the disease incidence was measured and the results showed that all de CFS-PG had a preventive action, being the CFS-PG of FZB42 the most active. Our work showed that the FZB42, CA1 and BQST713 and their metabolites evaluated in this study could be a new technology to be used as natural alternatives to synthetic fungicides in the control of postharvest diseases on lemons.