Respiratory inhibitors are among the fungicides most widely used for disease control on crops. Most are strobilurins and carboxamides, inhibiting the cytochrome b of mitochondrial complex III and the succinate dehydrogenase of mitochondrial complex II, respectively. A few years after the approval of these inhibitors for use on grapevines, field isolates of Botrytis cinerea , the causal agent of gray mold, resistant to one or both of these classes of fungicide were recovered in France and Germany. However, little was known about the mechanisms underlying this resistance in field populations of this fungus. Such knowledge could facilitate resistance risk assessment. The aim of this study was to investigate the mechanisms of resistance occurring in B. cinerea populations. Highly specific resistance to strobilurins was correlated with a single mutation of the cytb target gene. Changes in its intronic structure may also have occurred due to an evolutionary process controlling selection for resistance. Specific resistance to carboxamides was identified for six phenotypes, with various patterns of resistance levels and cross-resistance. Several mutations specific to B. cinerea were identified within the sdhB and sdhD genes encoding the iron-sulfur protein and an anchor protein of the succinate dehydrogenase complex. Another as-yet-uncharacterized mechanism of resistance was also recorded. In addition to target site resistance mechanisms, multidrug resistance, linked to the overexpression of membrane transporters, was identified in strains with low to moderate resistance to several respiratory inhibitors. This diversity of resistance mechanisms makes resistance management difficult and must be taken into account when developing strategies for Botrytis control.

Botrytis cinerea is a major crop pathogen infesting >220 hosts worldwide. A cryptic species has been identified in some French populations but the new species, B. pseudocinerea, has not been fully delimited and established. The aim of this study was to distinguish between the two species, using phylogenetic, biological, morphological, and ecological criteria. Multiple gene genealogies confirmed that the two species belonged to different, well-supported phylogenetic clades. None of the morphological criteria tested (spore size, germination rate, or mycelial growth) was able to discriminate between these two species. Sexual crosses between individuals from the same species and different species were carried out. Only crosses between individuals from the same species were successful. Moreover, population genetics analysis revealed a high level of diversity within each species and a lack of gene flow between them. Finally, a population survey over time showed that B. cinerea was the predominant species but that B. pseudocinerea was more abundant in spring, on floral debris. This observation could not be explained by temperature adaptation in tests carried out in vitro or by aggressiveness on tomato or bean leaves. This study clearly establishes that B. cinerea and B. pseudocinerea constitute a complex of two cryptic species living in sympatry on several hosts, including grapevine and blackberry. We propose several biological or molecular tools for unambiguous differentiation between the two species. B. pseudocinerea probably makes a negligible contribution to gray mold epidemics on grapevine. This new species has been deposited in the MycoBank international database.