In vitro toxicity of alternative oxidase inhibitors salicylhydroxamic acid and propyl gallate on Fusicladium effusum

Abstract

Pecan scab, caused by the fungus Fusicladium effusum (syn. Cladosporium caryigenum and Fusicladosporium effusum), is the most devastating disease of the commercial pecan (Carya illinoinensis) production in Southeastern United States. Disease control depends primarily on multiple applications of fungicides per season. Fungicides available for scab control include those in the quinone outside inhibitor (QoI) group. QoI fungicides have a high level of resistance risk and in vitro sensitivity assays are essential to monitor development of resistance in populations of F. effusum. In vitro assays were conducted to determine sensitivity of F. effusum to the QoI fungicide azoxystrobin. In vitro assays were conducted with and without the addition of salicylhydroxamic acid (SHAM) to inhibit alternative oxidase (AOX) and eliminate the rescue impact of AOX on fungal respiration in the presence of QoI fungicides. When 0.653 mM SHAM was added to the medium, growth of a majority of the isolates was significantly reduced, even in the absence of fungicide. Of the 89 isolates tested, only 21 showed a dose response to azoxystrobin. Growth of the other 68 isolates was insufficient to measure a dose response. Only 18 isolates showed a clear dose response to azoxystrobin both with and without SHAM, but the calculated EC50 values were significantly different (P < 0.0001). Further experiments were performed to investigate the effects of different concentrations of SHAM and another AOX inhibitor, propyl gallate (PG) on hyphal growth derived from mycelial fragments of F. effusum in liquid medium in microtiter plates and hyphal growth derived from germinated conidia on solid medium in the absence of fungicide. Even at the lowest concentrations tested, both SHAM and PG (0.01 and 0.25 mM, respectively) inhibited hyphal growth of F. effusum in liquid medium and on solid medium. These results suggest the role of AOX and possible auxiliary toxicity of these inhibitors in F. effusum physiology. Due to the toxic effects of these AOX inhibitors on F. effusum in vitro, future sensitivity profiling for QoIs will be performed without the addition of these inhibitors.