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. (

The efficacies of the new strobilurin fungicide kresoxim-methyl for the protection of apple leaves from infection by baseline populations of Venturia inaequalis were uniform across five major apple growing regions in North America. The mean ED50 value determined for 25 populations was 0.35 μg ml-1, with values ranging from 0.11 μg ml-1 to 0.75 μg ml-1. The mean level of scab control achieved at the kresoxim-methyl dose of 4 μg ml-1 was 93%. For one of the five orchards sampled in each region, kresoxim-methyl sensitivities of germinating conidia were determined. Sensitivities of 250 isolates were broadly distributed, with ED50 values ranging from 0.003 μg ml-1 to 0.14 μg ml-1 and a mean of 0.02 μg ml-1. This broad range of in vitro sensitivities was not reflected for the in vivo efficacy of kresoxim-methyl in the protection of apple leaves from scab infections. The discrepancy between in vivo and in vitro sensitivities implies that in vivo tests are more useful for the monitoring of kresoxim-methyl sensitivities of orchard populations. Because it can be expected that only isolates resistant under both test conditions will be prone to future selection, such isolates will contribute to increased frequencies of the least sensitive isolates described in this baseline study. Testing of in vitro isolate sensitivities will, therefore, provide an additional tool in the monitoring of kresoxim-methyl resistance development.