Inheritance and Mechanisms of Resistance to Anilinopyrimidine Fungicides in Botrytis Cinerea (Botryotinia Fuckeliana)

Monitoring conducted in French vineyards since 1994 led to detection of three anilinopyrimidine-resistant phenotypes in field populations of Botrytis cinerea (Botryotinia fuckeliana). A few strains were highly resistant to anilinopyrimidines (e.g., pyrimethanil) only and were named AniR1. Analysis of tetrads obtained from crosses between AniR1 and sensitive strains (AniS) showed that one major gene is probably involved in the AniR1 phenotype. The resistance mechanism has not yet been elucidated but the AniR1 strains did not seem to display a reduced accumulation of [14C]pyrimethanil in their mycelium. We also distinguished two different patterns of cross-resistance, named AniR2 and AniR3 phenotypes. Both were weakly resistant to anilinopyrimidines, highly resistant to tolnaftate, a squalene epoxidase inhibitor (used only in medicine), and appeared less sensitive to dicarboximides (e.g., iprodione). Moreover, AniR2 strains were weakly resistant to phenylpyrroles (e.g., fludioxonil) while AniR3 strains displayed a slight decrease of sensitivity to sterol C14demethylation inhibitors (e.g., prochloraz, tebuconazole) and to the experimental hydroxyanilide fenhexamid. Genetic analysis showed that there is no disjunction of multiple resistances in ascospore progenies and we found a 1:1 segregation of sensitive:resistant phenotypes, suggesting that AniR2 and AniR3 phenotypes are each probably encoded by a single major gene. Studies on the uptake of [14C]pyrimethanil did not show any difference between the sensitive and the resistant strains. However, the addition of fluazinam, an uncoupler, led to an accumulation of the fungicide in mycelium, suggesting that an active efflux occurred. Experiments carried out on the uptake of [14C]tebuconazole and [14C]triadimenol, two sterol C14-demethylation inhibitors, showed a lower accumulation of these fungicides in mycelium of resistant strains than in sensitive strains. The effect of fluazinam demonstrated that this phenomenon was correlated with an energy-dependent efflux, which seemed to be inducible in sensitive strains and constitutive in resistant strains and could be a multidrug resistance mechanism.