A specific antifungal activity and biofilm formation induced by the interaction between Bacillus subtilis and Setophoma terrestris

Córdoba

Congreso; XI CONGRESO ARGENTINO DE MICROBIOLOGÍA GENERAL SAMIGE; 2015

Onion is the second vegetable crop produced worldwide. Among the major limiting diseases of onion is pink root. Its causal agent, Setophoma terrestris, is one of the most severe pathogens. We decided to address the biocontrol as a mean to control th is main pathogen of onion in Argentina. We have previously isolated bacteria with antagonistic activity against S. terrestris from soil samples under continuous onion culture. The most efficient isolate was characterized by MALDI-TOF as Bacillus subtilis subsp. subtilis. The activity of B. subtilis against S. terrestris was screened by dual cultures and antagonism evaluated by measuring the radial growth of the pathogen after 3-4 days of incubation. B. subtilis showed a strong capacity of growth inhibition of S. terrestris. To assess whether the fungal inhibition was determined only by the release of diffusible extracellular bacterial metabolites, the antifungal activity of the cell-free supernatant of B. subtilis cultures were evaluated. Interestingly, we observed a high fungal growth inhibition on plates containing cell-free supernatant of B. subtilis previously grown in the presence of the fungus (BS post-St). In contrast, no significant differences in the fungal growth were obtained between control plates and plates containing cell-free supernatant from B. subtilis grown without previous contact with S. terrestris. Moreover, no antagonistic activity against other onion fungal pathogens such as Fusarium oxysporum was found, indicating a this activity was specific against S. terrestris. In fact, before bacteria were overtaken by the mycelium of F. oxysporum, a sample of B. subtilis from co-culture plates (BS post-Fo) was recovered and its antifungal activity of cell-free supernatant against S. terrestris was tested. We observed no significant differences between control plates and those containing cell-free supernatant from BS post-Fo. In addition, an enhance biofilm forming ability and an increment in pellicle formation in BS post-St compared to B. subtilis grown without previous contact with S. terrestris was observed. Consistently, BS post-St showed an inhibition of swarming motility. Finally, the antagonistic activity was also tested in interaction assays in onion plants cultivated in vitro. For plants not inoculated with B. subtilis we observed a loss of vigor of the roots and dark coloration of tips roots when infected with the fungus. On the other hand, no abnormalities were found in roots of plants inoculated with B. subtilis. Furthermore, we found a strong growth inhibition of S. terrestris, with no invasion of hypha inside the medium, and an increase of bacterial density, especially near the root system in tubes containing plants inoculated with B. subtilis. Taken together, our results suggest that B. subtilis subsp. subtilis releases diffusible bacterial inhibitory compounds whose secretion is specifically induced by the interaction with the fungal pathogen S. terrestris and that B. subtilis undergoes cell differentiation that results in biofilm formation after contact with the fungus.