Volatile self-inhibitor of spore germination in pathogenic Mucorale Rhizopus arrhizus

Rhizopus arrhizus is a common pathogenic Mucoralean mold that exists as a saprophyte, and is disseminated through sporangiospores, which germinate to form mycelia under suitable environmental or infection settings. Such morphological transitions are often mediated by self-produced effector molecules in a density-dependent fashion. This study aimed to elucidate if a quorum-dependent, cell-density-driven phenomenon exists in R. arrhizus, and identify the molecule(s) involved. The germination of R. arrhizus was observed to be reliant on the seeding density, with nearly 71% and 47% germination in Sabouraud dextrose and glucose asparagine media respectively at 1 × 105-1 × 106 spores/mL, and only 10% and 1% germination respectively with 1 × 108 spores/mL. The late-growth-stage supernatant also hindered the spore germination and liquid-culture biomass in a dose-dependent way. These effects were being mediated by a volatile inhibitor present in the headspace and supernatant of R. arrhizus cultures, identified as 2-methyl-2-butene by gas chromatography and electron ionization-quadrupole mass spectrometry. The compound was present in a density-dependent manner and considerably impaired fungal germ-tube emergence and elongation during germination. Spore swelling remained unaffected. Multiple thin protrusions comprising of F-actin and microtubules were seen emanating from the treated cells, suggestive of filopodia-like and cytoneme-like extensions. The same compound was also detected in Rhizomucor pusillus.