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Appl. Environ. Microbiol., Jan 1997, 178-185, Vol 63, No. 1
Copyright © 1997, American Society for Microbiology

Nonanoic Acid, a Fungal Self-Inhibitor, Prevents Germination of Rhizopus oligosporus Sporangiospores by Dissipation of the pH Gradient

P Breeuwer, JC De Reu, J Drocourt, FM Rombouts and T Abee
Department of Food Science, Wageningen Agricultural University, 6703 HD, Wageningen, The Netherlands, and Chemunex S.A., 94700 Maisons-Albort, France

Germination of Rhizopus oligosporus sporangiospores is characterized by swelling of the spores and subsequent emergence of germ tubes. Changes in spore morphology and alterations in intracellular pH (pH(infin)) of the sporangiospores were assessed during the germination process by flow cytometry. Sporangiospores were stained with carboxyfluorescein by incubation with carboxyfluorescein diacetate. The nonfluorescent carboxyfluorescein diacetate is passively transported into intact cells and subsequently cleaved by esterases, which results in intracellular accumulation of the fluorescent carboxyfluorescein. Given that the fluorescence of carboxyfluorescein is pH dependent, the pH(infin) of the individual spores could be assessed simultaneously with spore size. For R. oligosporus, swelling of the sporangiospores was accompanied by an increase of pH(infin). In the presence of nonanoic acid, a self-inhibitor produced by various fungi, increase of the pH(infin) was prevented and swelling was inhibited at concentrations of less than 1 mM. Octanoic acid and decanoic acid were equally effective. Acetic acid also inhibited germination but at much higher concentrations (>8 mM). The mechanism of action of these fatty acids is most likely dissipation of the pH gradient. A model is proposed in which the pH(infin) plays a crucial role in the germination of R. oligosporus sporangiospores.