FIG 3.

Culture extracts from symbiotic Rhizopus microsporus kills Protostelium aurantium. (A) The survival of P. aurantium, indicated by the diameter of the predation plaque (clearance of yeast), is significantly reduced in cultures that were exposed to 2% crude culture extract from symbiotic R. microsporus (RMsym). Incubation with solvent alone (DMSO) or apo-symbiotic R. microsporus (RMapo) has no effect on the viability of P. aurantium. Circles indicate independent replicated experiments (n = 3) ± 1 SEM (gray bars). One-way ANOVA with Tukey’s multiple-comparison test was performed (*, P < 0.0001; see Table S2). (B) Photographs of yeast agar plates showing the predation plaque by P. aurantium (arrowheads). (C) HPLC profiles of crude extracts from symbiotic and endosymbiont-free R. microsporus showing a mixture of rhizoxin derivatives, including the two major bacterial rhizoxin congeners (rhizoxin S1 and rhizoxin S2). The peak correlating to rhizoxin is marked with an asterisk (*). Monitoring was done at 310 nm (see Fig. S2). The peak areas were integrated to calculate the concentration of rhizoxin S1 (1.2 μM) and rhizoxin S2 (1.7 μM), as well as all rhizoxin congeners combined (8.7 μM). (D) Fluorescence microscopy images of symbiotic R. microsporus and endosymbiont-free R. microsporus. Green fluorescence indicates presence of endosymbionts (SYTO9). Scale bars, 5 μm.