Investigating the use of natural products as potential antifungals

Rhizopus microsporusis a spore-forming filamentous fungus of the order Mucorales. Rhizopus species, are responsible for post-harvest rot of many soft fruits and vegetables, and are one of the aetiological agents causing the fatal disease mucormycosis. Given that Rhizopus spp. are naturally resistant to several commercial antifungal therapies, it is important that we find novel approaches to control the growth of these fungi. Here we investigate the antifungal activity ofsecreted metabolites from the Gram-negative bacterium Pseudomonas aeruginosa and the plant derived compound madecassic acid from the medicinal herb Centella asiatica, against R. microsporus. Both P. aeruginosa (PA01 and PA14) cells and supernatants displayed significant antifungal activity, killing 96% of the fungal spores, suggest that the bacterium secretes a molecule(s) with significant antifungal potential. Although P. aeruginosa secreted siderophores inhibited fungal growth via iron chelation, these siderophores were fungistatic, not fungicidal in the PA14 strain. Screening bacterial mutants in key secondary metabolites including phenazines, rhamnolipids, quinolones and polyhydroxyalkanoates, did not relieve the antifungal activity of the bacterium, suggesting these key virulence factors are not required for this effect. Pre-incubation of supernatant with spores before use as treatment against fresh spores showed removal of antifungal activity, suggesting that the fungus might metabolise or sequester the antifungal molecule. The plant-based molecule, madecassic acid exhibited antifungal activity against R. microsporus, Candida albicans and Cryptococcus neoformans with concentrations of 250 µM inhibiting growth for all three fungi. Our findings show that both P. aeruginosa and madecassic acid have potential for use as antifungal treatments alternative to those currently commercially available.