The class of Qo-inhibiting fungicides (QoIs) act as respiration inhibitors by binding to the Qo center of cytochrome b. The longevity of these fungicides has been challenged by the selection of fungal sub-populations resisting high doses of QoI fungicides, with a G143A amino acid exchange in the cytochrome b target site identified as the most common cause of resistance. In contrast, the mechanism of alternative respiration, as another mechanism of fungal QoI resistance, has thus far not been affiliated with practical resistance. In the present study, azoxystrobin-resistant mutants of Magnaporthe grisea were generated and characterized. Emergence of these spontaneous mutants was facilitated when resting melanized mycelia were allowed to escape full inhibition by azoxystrobin. This escape was related to the intactness of alternative respiration, indicating that residual expression of this rescue mechanism was involved in the spontaneous emergence of target-site mutants. The two mutants characterized resisted high doses of the QoI, azoxystrobin, with resistance factors exceeding 1,000. Two different mutations of the cytochrome b gene were identified as exchanges of guanine, leading to a G143A or a G143S amino acid exchange. Resistance of both target-site mutants remained stable during four consecutive disease cycles in the absence of azoxystrobin. Several parameters tested to measure fitness penalties inherent to the mutational changes revealed that the G143A mutant was not compromised. In contrast, the conidia production of the G143S mutant was significantly lower under both saprophytic and pathogenic conditions of reproduction.

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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.

Monoconidial isolates of Venturia inaequalis were collected in 1990 and 1991 from orchards in New York, Michigan, and Nova Scotia that had never or only sporadically been treated with fungicides acting as sterol demethylation inhibitors (DMIs). Sensitivities of isolates to two representative DMIs (fenarimol and myclobutanil) were determined by a sensitivity test based on the relative growth (RG) of mycelial colonies at one discriminatory dose. Mean isolate sensitivities were not significantly different (P > 0.2) for the majority of the populations tested, and all sensitivity data obtained from these sites were combined to provide a baseline distribution of isolate sensitivities for both fenarimol and myclobutanil. The baseline distributions were compared with isolate sensitivities determined for an experimental orchard in Nova Scotia with a documented case of DMI resistance and for a commercial orchard in Michigan with a long history of DMI use and first evidence of practical DMI resistance. For both DMIs tested and in both treated orchards, frequencies of isolates with RG values <80 had decreased or only slightly increased compared to the baseline population. In contrast, frequencies of isolates with RG values >80 had increased more than 20-fold over baseline levels. Thus, isolates with RG values >80 were rated DMI resistant. The validity of a qualitative isolate classification was tested in controlled infection studies. At doses of fenarimol and myclobutanil recommended for commercial control of apple scab, reproduction of a typical sensitive isolate on treated apple leaves was suppressed completely. Lesions caused by a resistant isolate continued to expand and produced abundant conidia. Statistical analysis of orchard sensitivities revealed that the analysis of isolate counts grouped into the categories DMI sensitive or resistant was most indicative in comparisons of orchard sensitivities aimed at detection of practical DMI resistance. A high degree of cross-resistance between fenarimol and myclobutanil indicated that sensitivity tests with one of the DMIs employed as the diagnostic tool in this study can serve as a test for other DMIs.