CHARACTERIZATION OF THE IMPACT OF BACILLUS LICHENIFORMIS AND PSEUDOMONAS AERUGINOSA AGAINST ALTERNARIA ALTERNATA BY PHASE CONTRAST MICROSCOPY AND TRANSMISSION ELECTRON MICROSCOPY

Pseudomonas and Bacillus are the most studied groups of plant disease-suppressive bacteria. Plant protection is mediated by the antagonistic effects of these bacteria against phytopathogens. The antifungal activity of several Pseudomonas spp. with highly degrading capacities, used in soil bioremediation, respectively some strains derived from Bacillus licheniformis, was established by dual test cultures with different fungal pathogen species as Alternaria alternata, Pythium debaryanum, Fusarium oxysporum and Botrytis cinerea. In early stages, Pseudomonas strains have a higher inhibitory activity on mycelium development, although the inhibition zone is more stable in time for Bacillus licheniformis. For the microscopic analyses, liquid media inoculated with Alternaria alternata, and simple or mixed culture of bacilli respectively pseudomonads were used. Light microscopy observations showed that pseudomonads tend to adhere along hyphae, while bacilli form clusters around hyphae. Transmission electron micrographs revealed that in interaction with bacilli, fungal cells release numerous electron-dense granules or vesicles with fibrilar content, while in the presence of pseudomonads, the fungal cell walls were coated by a network of microfilaments and fibrils. Most fungal cells have corrugated, very thick and heavily melanized cell walls, characteristic to the spores and are spatially separated by bacilli. In interaction with pseudomonads, fungal cells were surrounded by bacteria and presented detachment of the plasmatic membranes, coalescence of cytoplasm and degradation of intracellular membrane system. The destruction and disorganization of cell content in hyphae and conidia was also observed in the later stage of coculture with bacilli. The results indicate that the two types of bacteria have different mechanisms of action against fungal pathogen.