1998). The respiratory inhibition caused by QoI fungicides is believed to involve proton pooling, which leads to the production of reactive oxygen species (ROS). Once the metabolic activity is inhibited, the ROS generated may activate AOX and restart germination. The AOX pathway is also PD0332991 considered to have a protective role against oxygen stress (Maxwell et al., 1999; Magnani et al., 2008; Van Aken et al., 2009). The AOX pathway can be inhibited by salicylhydroxamic acid (SHAM) or n-propyl gallate (PG) (Siedow & Bickett, 1981). If the electron flow in the respiratory chain is interrupted, excessive electrons are pooled. Under such circumstances, excessive electrons can cause aberrant generation of ROS (Kim et al., 2008). Indeed, in Fusarium graminearum, treatment with azoxystrobin (AZ) induced ROS production and AOX induction, and treatment with AZ + SHAM generated additional ROS compared to AZ treatment alone (Kaneko & Ishii, 2009). Moreover, the quantity of ROS generation and AOX activity were correlated with AZ sensitivity between F. graminearum and Microdochium nivale (Kaneko & Ishii, 2009). Excessive ROS generation may cause death at the beginning of mitochondrial destruction. In Penicillium digitatum, oxidative stress produced by exogenous treatment with hydrogen peroxide caused ultrastructural
disorganization (Cerioni selleck et al., 2010). Moreover, in Aspergillus nidulans and yeast, farnesol-induced apoptosis participated in mitochondrial generation of ROS (Machida et al., 1998; Semighini et al., 2006). In Botrytis cinerea, the presence of dead cells following treatment with AZ and PG was confirmed by vital indicator, calcein-AM (acetoxymethyl ester), and nucleus staining (Takahashi et al., 2008). In this experiment, however, the cell death was evaluated after a long incubation period (3 or 4 days). Therefore,
it was not clear whether the fatal effect was directly caused by AZ and PG. In contrast, in another phytopathogenic fungus, Mycosphaerella graminicola, the effect of AZ was found to be fungistatic on the host plant (Rohel et al., 2001). In this study, we evaluated the effect of AZ and AOX inhibitors on the spore germination of the grey mould fungus, B. cinerea, by cytological analyses. Botrytis cinerea isolate almost from strawberry IBA1-2-1 (AZ-sensitive) (Ishii et al., 2009) was used. To promote sporulation, three mycelial plugs were inoculated on PDA (Becton Dickinson, Franklin Lakes, NJ) in a 9-cm Petri dish, incubated for 3 days at 20 °C under darkness, for 4 days at 20 °C under near UV irradiation, and then for another 3 days at 20 °C under darkness. The aerial hyphae-bearing conidia were washed with distilled water (DW), rubbed off the media with a paintbrush, and filtered through a Kimwipe S-200 (Cresia Corp., Tokyo, Japan) to remove the hyphal fragments.