Quinone outside inhibiting (QoI) fungicides represent one of the most widely used groups of fungicides used to control agriculturally important fungal pathogens. They inhibit the cytochrome bc 1 complex of mitochondrial respiration. Soon after their introduction onto the market in 1996, QoI fungicide-resistant isolates were detected in field plant pathogen populations of a large range of species. However, there is still little understanding of the processes driving the development of QoI fungicide resistance in plant pathogens. In particular, it is unknown whether fungicide resistance occurs independently in isolated populations or if it appears once and then spreads globally by migration. Here, we provide the first case study of the evolutionary processes that lead to the emergence of QoI fungicide resistance in the plant pathogen Plasmopara viticola. Sequence analysis of the complete cytochrome b gene showed that all resistant isolates carried a mutation resulting in the replacement of glycine by alanine at codon 143 (G143A). Phylogenetic analysis of a large mitochondrial DNA fragment including the cytochrome b gene (2,281 bp) across a wide range of European P. viticola isolates allowed the detection of four major haplotypes belonging to two distinct clades, each of which contains a different QoI fungicide resistance allele. This is the first demonstration that a selected substitution conferring resistance to a fungicide has occurred several times in a plant-pathogen system. Finally, a high population structure was found when the frequency of QoI fungicide resistance haplotypes was assessed in 17 French vineyards, indicating that pathogen populations might be under strong directional selection for local adaptation to fungicide pressure.Preventing and delaying resistance adaptation to fungicides in pathogen populations is a major goal in sustainable plant pathogen management. The development of effective strategies to control pathogen populations requires precise understanding of the conditions under which fungicide resistance alleles appear, spread, and are maintained in natural populations. Among the factors that influence the evolution of resistance, the rate of mutation in fungicide resistance-associated genes plays an important role. It is still uncertain whether fungicide resistance-associated mutations commonly arise independently or whether pest adaptation occurs by convergent evolution through recurrent mutations at selected sites in the genes coding for the fungicide target. In other words, do alleles conferring fungicide resistance arise once before spreading across populations, or do they arise independently in each locality, and are they favored by large local population sizes of the pathogen population?Numerous experimental and phylogenetic studies in extensive geographical contexts have already examined the pattern of the evolution of genes coding for antimicrobial drugs or pesticide targets in animals, plants, and bacteria. For instance, the question of parallel genetic adaptation to drugs and p...

We investigated the molecular basis of resistance of the obligate biotrophic grape powdery mildew fungus Uncinula necator to sterol demethylation-inhibiting fungicides (DMIs). The sensitivity of 91 single-spore field isolates of U. necator to triadimenol was assessed by using a leaf disc assay. Resistance factors (RF) ranged from 1.8 to 26.0. The gene encoding the target of DMIs (eburicol 14␣-demethylase) from five sensitive and seven resistant isolates was cloned and sequenced. A single mutation, leading to the substitution of a phenylalanine residue for a tyrosine residue at position 136, was found in all isolates exhibiting an RF higher than 5. No mutation was found in sensitive or weakly resistant (RF, <5) isolates. An allele-specific PCR assay was developed to detect the mutation. Among the 91 isolates tested, only isolates with RF higher than 5 carried the mutation. Three of the 19 resistant isolates and all sensitive and weakly resistant isolates did not possess the mutation. The mutation at codon 136 is thus clearly associated with high levels of resistance to triadimenol.

Molecular studies of some micro-organisms are hampered by the difficulty of obtaining sufficient amounts of nucleic acids. A cloning strategy based on PCR has therefore been used to clone the eburicol 14alpha-demethylase (CYP51) gene of the obligate fungus Erysiphe graminis f. sp. hordei (Egh) using minute amounts of genomic DNA. The CYP51 gene encodes the enzymatic target of a major group of fungicides. Sequencing CYP51 from different Egh isolates revealed the occurrence of two alleles for this gene. An allele-specific PCR assay was developed to detect each CYP51 allele.

Stilbene-enriched extracts from Vitis vinifera waste (cane, wood, and root) were characterized by UHPLC-MS. Eleven stilbenes were identified and quantified as follows: ampelopsin A, (E)-piceatannol, pallidol, (E)-resveratrol, hopeaphenol, isohopeaphenol, (E)-ε-viniferin, (E)-miyabenol C, (E)-ω-viniferin, r2-viniferin, and r-viniferin. The fungicide concentration inhibiting 50% of growth of Plasmopara viticola sporulation (IC) was determined for the extracts and also for the main compounds isolated. r-Viniferin followed by hopeaphenol and r2-viniferin showed low IC and thus high efficacy against Plasmopara viticola. Regarding stilbene extracts, wood extract followed by root extract showed the highest antifungal activities. These data suggest that stilbene complex mixtures from Vitis vinifera waste could be used as a cheap source of bioactive stilbenes for the development of natural fungicides.