The Indigenous Volatile Inhibitor 2-Methyl-2-butene Impacts Biofilm Formation and Interspecies Interaction of the Pathogenic Mucorale Rhizopus arrhizus.

2-Methyl-2-butene has recently been reported to be a quorum-based volatile self-inhibitor of spore germination and growth in pathogenic Mucorale Rhizopus arrhizus. The present study aimed to elucidate if this compound can influence R. arrhizus biofilm formation and interspecies interaction. The compound was found to significantly decrease R. arrhizus biofilm formation (p < 0.001), with nearly 25% and 50% lesser biomass in the biofilms cultured with exposure to 4 and 32 µg/ml of 2-methyl-2-butene, respectively. The growth of pre-formed biofilms was also impacted, albeit to a lesser extent. Additionally, 2-methyl-2-butene was found to self-limit R. arrhizus growth during interspecies interaction with Staphylococcus aureus and was detected at a substantially greater concentration in the headspace of co-cultures (2338.75 µg/ml) compared with monocultures (69.52 µg/ml). Some of the C5 derivatives of this compound (3-methyl-1-butanol, 2-methyl-2-butanol, and 3-methyl-1-butyne) were also observed to partially mimic its action, such as inhibition of spore germination, but did not impact R. arrhizus biofilm formation. Finally, the treated R. arrhizus displayed changes in fungal morphology suggestive of cytoskeletal alterations, such as filopodia formation, blebs, increased longitudinal folds and/or corrugations, and finger-like and sheet-like surface protrusions, depending upon the concentration of the compound(s) and the planktonic or biofilm growth mode.