Several agricultural fungicides related to the antifungal strobilurins act as inhibitors of respiration by binding to mitochondrial cytochrome b. Two types of laboratory mutants resisting higher doses of the strobilurin-related inhibitor kresoxim-methyl were characterized for Venturia inaequalis, the causal agent of apple scab. Selection of mutagenized conidia by kresoxim-methyl yielded mutants altered in the expression of alternative respiration during the stage of conidia germination. Cytochrome b sequences were not affected in the respective mutants. Selection of conidia on media containing the alternative oxidase inhibitor salicylhydroxamic acid in addition to kresoxim-methyl yielded a highly resistant mutant distinguished by a G143A exchange in cytochrome b. The status of mitochondrial cytochrome b genes remained heteroplasmic, and mitochondria containing wild-type cytochrome b returned to high frequencies during cultivation on inhibitor-free medium. However, continuation of the selection process led to a more pronounced replacement of sensitive by mutated mitochondria. The G143A mutation of cytochrome b causing resistance of V. inaequalis to a strobilurin-related inhibitor has been reported previously for mouse mitochondria; and a permanent G143A exchange rendering naturally resistant mitochondria has been reported for the strobilurin-producing basidiomycete Mycena galopoda and for the sea urchin Paracentrotus lividus. At the corresponding position, alanine was also present in chloroplast cytochrome b6 exhibiting low binding of strobilurin-related inhibitors. The mutation of cytochrome b reported here for V. inaequalis describes the first example of a mutation in filamentous ascomycetes and is part of an assessment of resistance risks inherent to strobilurin fungicides.

: A new class of agricultural fungicides derived from strobilurins act as respiration inhibitors by binding to mitochondrial cytochrome b. The e †ects of the strobilurin, kresoxim-methyl, on conidia germination, mycelial growth and the protection of apple leaves from scab development were investigated for two isolates of V enturia inaequalis randomly selected from a culture collection. Inhibition of mycelial growth required relatively high doses of kresoxim-methyl for both isolates. In comparison, germination of conidia was (ED 50 \ 1 lg ml~1) highly sensitive for one of the isolates while the level of (ED 50 \ 0É005 lg ml~1), inhibition achieved for the second isolate was 60-fold less (ED 50 \ 0É3 lg ml~1). As deduced from identical sequences of cytochrome b cDNAs prepared from both isolates, the di †erent responses of germinating conidia to kresoxim-methyl were not caused by di †erences in the sequence of cytochrome b as the target site for strobilurin action. Strong synergistic e †ects of salicylhydroxamic acid on kresoxim-methyl inhibitory potency suggested that the reduced kresoxim-methyl sensitivity observed for germinating conidia was caused by interference of the alternative respiratory pathway with inhibitor action. However, this mechanism of target site circumvention in germinating conidia had no adverse e †ects on the protection of apple leaves from scab infection by kresoxim-methyl. (

A new class of agricultural fungicides derived from the group of antifungal strobilurins acts as specific respiration inhibitors by binding to mitochondrial cytochrome b. The cytochrome b gene was cloned and sequenced from the mitochondrial genome of Venturia inaequalis, the causal agent of apple scab. The gene was 10.65 kbp in size and contained seven exons and six introns. The exons encoded a protein of 393 amino acids. Comparison of the deduced amino-acid sequence with cytochrome b proteins from other fungi revealed highest homologies to the respective proteins of Aspergillus nidulans, Podospora anserina and Neurospora crassa. All amino acids of the V. inaequalis cytochrome b at positions altered in mutants of Saccharomyces cerevisiae resistant to strobilurins, and other fungi with reduced sensitivities to strobilurins, were identical to wild-type isolates of several fungi. The cloning and characterization of the V. inaequalis cytochrome b gene is the initial step in the assessment of resistance risks inherent to the strobilurin fungicides.