The new broad-spectrum fungicides from the succinate dehydrogenase inhibitor (SDHI) class have been quickly adopted by the market, which may lead to a high selection pressure on various pathogens. Cases of resistance have been observed in 14 fungal pathogens to date and are caused by different mutations in genes encoding the molecular target of SDHIs, which is the mitochondrial succinate dehydrogenase (SDH) enzyme. All of the 17 marketed SDHI fungicides bind to the same ubiquinone binding site of the SDH enzyme. Their primary biochemical mode of action is the blockage of the TCA cycle at the level of succinate to fumarate oxidation, leading to an inhibition of respiration. Homology models and docking simulations explain binding behaviors and some peculiarities of the cross-resistance profiles displayed by different members of this class of fungicides. Furthermore, cross-resistance patterns among SDHIs is complex because many mutations confer full cross resistance while others do not. The nature of the mutations found in pathogen populations varies with species and the selection compound used but cross resistance between all SDHIs has to be assumed at the population level. In most of the cases where resistance has been reported, the frequency is still too low to impact field performance. However, the Fungicide Resistance Action Committee has developed resistance management recommendations for pathogens of different crops in order to reduce the risk for resistance development to this class of fungicides. These recommendations include preventative usage, mixture with partner fungicides active against the current pathogen population, alternation in the mode of action of products used in a spray program, and limitations in the total number of applications per season or per crop.

The gray mold fungus Botrytis cinerea is a major threat to fruit and vegetable production. Strawberry fields usually receive several fungicide treatments against Botrytis per season. Gray mold isolates from several German strawberry-growing regions were analyzed to determine their sensitivity against botryticides. Fungicide resistance was commonly observed, with many isolates possessing resistance to multiple (up to six) fungicides. A stronger variant of the previously described multidrug resistance (MDR) phenotype MDR1, called MDR1h, was found to be widely distributed, conferring increased partial resistance to two important botryticides, cyprodinil and fludioxonil. A 3-bp deletion mutation in a transcription factor-encoding gene, mrr1, was found to be correlated with MDR1h. All MDR1h isolates and the majority of isolates with resistance to multiple fungicides were found to be genetically distinct. Multiple-gene sequencing confirmed that they belong to a novel clade, called Botrytis group S, which is closely related to B. cinerea and the host-specific species B. fabae. Isolates of Botrytis group S genotypes were found to be widespread in all German strawberry-growing regions but almost absent from vineyards. Our data indicate a clear subdivision of gray mold populations, which are differentially distributed according to their host preference and adaptation to chemical treatments.

Following earlier incomplete and fragmented versions of a genome sequence for the grey mould Botrytis cinerea, a gapless, near-finished genome sequence for B. cinerea strain B05.10 is reported. The assembly comprised 18 chromosomes and was confirmed by an optical map and a genetic map based on approximately 75 000 single nucleotide polymorphism (SNP) markers. All chromosomes contained fully assembled centromeric regions, and 10 chromosomes had telomeres on both ends. The genetic map consisted of 4153 cM and a comparison of the genetic distances with the physical distances identified 40 recombination hotspots. The linkage map also identified two mutations, located in the previously described genes Bos1 and BcsdhB, that conferred resistance to the fungicides boscalid and iprodione. The genome was predicted to encode 11 701 proteins. RNAseq data from >20 different samples were used to validate and improve gene models. Manual curation of chromosome 1 revealed interesting features, such as the occurrence of a dicistronic transcript and fully overlapping genes in opposite orientations, as well as many spliced antisense transcripts. Manual curation also revealed that the untranslated regions (UTRs) of genes can be complex and long, with many UTRs exceeding lengths of 1 kb and possessing multiple introns. Community annotation is in progress.

A range of novel carboxamide fungicides, inhibitors of the succinate dehydrogenase enzyme (SDH, EC 1.3.5.1) is currently being introduced to the crop protection market. The aim of this study was to explore the impact of structurally distinct carboxamides on target site resistance development and to assess possible impact on fitness.We used a UV mutagenesis approach in Mycosphaerella graminicola, a key pathogen of wheat to compare the nature, frequencies and impact of target mutations towards five subclasses of carboxamides. From this screen we identified 27 amino acid substitutions occurring at 18 different positions on the 3 subunits constituting the ubiquinone binding (Qp) site of the enzyme. The nature of substitutions and cross resistance profiles indicated significant differences in the binding interaction to the enzyme across the different inhibitors. Pharmacophore elucidation followed by docking studies in a tridimensional SDH model allowed us to propose rational hypotheses explaining some of the differential behaviors for the first time. Interestingly all the characterized substitutions had a negative impact on enzyme efficiency, however very low levels of enzyme activity appeared to be sufficient for cell survival. In order to explore the impact of mutations on pathogen fitness in vivo and in planta, homologous recombinants were generated for a selection of mutation types. In vivo, in contrast to previous studies performed in yeast and other organisms, SDH mutations did not result in a major increase of reactive oxygen species levels and did not display any significant fitness penalty. However, a number of Qp site mutations affecting enzyme efficiency were shown to have a biological impact in planta.Using the combined approaches described here, we have significantly improved our understanding of possible resistance mechanisms to carboxamides and performed preliminary fitness penalty assessment in an economically important plant pathogen years ahead of possible resistance development in the field.

Black Sigatoka or black leaf streak disease, caused by the Dothideomycete fungus Pseudocercospora fijiensis (previously: Mycosphaerella fijiensis), is the most significant foliar disease of banana worldwide. Due to the lack of effective host resistance, management of this disease requires frequent fungicide applications, which greatly increase the economic and environmental costs to produce banana. Weekly applications in most banana plantations lead to rapid evolution of fungicide-resistant strains within populations causing disease-control failures throughout the world. Given its extremely high economic importance, two strains of P. fijiensis were sequenced and assembled with the aid of a new genetic linkage map. The 74-Mb genome of P. fijiensis is massively expanded by LTR retrotransposons, making it the largest genome within the Dothideomycetes. Melting-curve assays suggest that the genomes of two closely related members of the Sigatoka disease complex, P. eumusae and P. musae, also are expanded. Electrophoretic karyotyping and analyses of molecular markers in P. fijiensis field populations showed chromosome-length polymorphisms and high genetic diversity. Genetic differentiation was also detected using neutral markers, suggesting strong selection with limited gene flow at the studied geographic scale. Frequencies of fungicide resistance in fungicide-treated plantations were much higher than those in untreated wild-type P. fijiensis populations. A homologue of the Cladosporium fulvum Avr4 effector, PfAvr4, was identified in the P. fijiensis genome. Infiltration of the purified PfAVR4 protein into leaves of the resistant banana variety Calcutta 4 resulted in a hypersensitive-like response. This result suggests that Calcutta 4 could carry an unknown resistance gene recognizing PfAVR4. Besides adding to our understanding of the overall Dothideomycete genome structures, the P. fijiensis genome will aid in developing fungicide treatment schedules to combat this pathogen and in improving the efficiency of banana breeding programs.