By contrast, ethanol-treated spores as a control for dead cells showed severely damaged plasma membranes and mitochondria (Fig. 4c). In the specimen treated with AZ and SHAM for 4 days, a lack of cristae and membrane breakage were observed in the mitochondria, but the frequency of this effect was very low, i.e. 23 of 264 mitochondria (in 6 of 38 spores; Fig. 4f and g). It was not easy to make conclusive judgements using chemical indicators as to whether the cells treated with AZ and AOX inhibitors were alive or dead. In the case of the trypan blue application, spores with positive signals were judged to

be dead cells. Indeed, the spores treated with 70% ethanol, as a dead control, showed positive signals. However, positive signals of trypan blue were also observed in the cells treated with DW, DMSO, AZ and SHAM, which had no apparent effect on spore germination. They

were stained at the cell membrane. It was Ibrutinib in vivo possible PS-341 concentration that the trypan blue dyes could be absorbed through active endocytosis at the hyphal tip during spore germination (Atkinson et al., 2002). The trypan blue dye is known to bind extracellular protein such as serum (Black & Berenbaum, 1964). The dye might bind to the extracellular matrix protein of B. cinerea germlings (Doss et al., 1995). Based on the results with these chemical indicators, we conclude that the spores treated with AZ and the AOX inhibitors are alive. Considering these issues, we performed other experiments to achieve our objective. The elimination of AZ and SHAM enabled the spore to germinate, suggesting that the effects of AZ and SHAM are reversible. Conversely, longer incubation (for more than 3 days) with AZ and SHAM decreased the spore germination rate, Guanylate cyclase 2C suggesting that a substantial portion of the spores were dead. Ultrastructural analysis also revealed that the treatments with AZ and SHAM were ineffective. We observed intact mitochondria, suggesting that the ROS generation in the treatment with AZ

and SHAM is not enough to affect mitochondrial integrity. The B. cinerea fungus is also known to resist oxidative stress (Gil-ad & Mayer, 1999). This result was supported by the reversibility of the effects of AZ and SHAM in the elimination experiment. Furthermore, longer incubation with AZ and SHAM appeared to cause mitochondrial destruction (although this effect was observed only with a low frequency) and death. However, it was difficult to conclude whether the fatality caused by longer incubation was due to a direct effect of AZ and AOX inhibitor or an indirect effect such as a metabolic disorder or autolysis. It has also been reported that a high concentration of carboxin is fungistatic, but a low concentration of carboxin is fungicidal in Ustilago nuda (Newcombe & Thomas, 1990). Carboxin probably has fungicidal effects only when the minimum amount of energy required for autolysis is available.