A dispensable paralog of succinate dehydrogenase subunit C mediates standing resistance towards a subclass of SDHI fungicides in Zymoseptoria tritici.

Succinate dehydrogenase inhibitor (SDHI) fungicides are widely used for the control of a broad range of fungal diseases. This has been the most rapidly expanding fungicide group in terms of new molecules discovered and introduced for agricultural use over the past fifteen years. A particular pattern of differential sensitivity (resistance) to the stretched heterocycle amide SDHIs (SHA-SDHIs), a subclass of chemically-related SDHIs, was observed in naïve Zymoseptoria tritici populations not previously exposed to these chemicals. Subclass-specific resistance was confirmed at the enzyme level but did not correlate with the genotypes of the succinate dehydrogenase (SDH) encoding genes. Mapping and characterization of the molecular mechanisms responsible for standing SHA-SDHI resistance in natural field isolates identified a gene paralog of SDHC, termed ZtSDHC3, which encodes for an alternative C subunit of succinate dehydrogenase, named alt-SDHC. Using reverse genetics, we showed that alt-SDHC associates with the three other SDH subunits, leading to a fully functional enzyme and that a unique Qp-site residue within the alt-SDHC protein confers SHA-SDHI resistance. Enzymatic assays, computational modelling and docking simulations for the two SQR enzymes (altC-SQR, WT_SQR) enabled us to describe enzyme-inhibitor interactions at an atomistic level and to propose rational explanations for differential potency and resistance across SHA-SDHIs. European Z. tritici populations displayed a presence (20-30%) / absence polymorphism of ZtSDHC3, as well as differences in ZtSDHC3 expression levels and splicing efficiency. These polymorphisms have a strong impact on SHA-SDHI resistance phenotypes. Characterization of the ZtSDHC3 promoter in European Z. tritici populations suggests that transposon insertions are associated with the strongest resistance phenotypes. These results establish that a dispensable paralogous gene determines SHA-SDHIs fungicide resistance in natural populations of Z. tritici. This study paves the way to an increased awareness of the role of fungicidal target paralogs in resistance to fungicides and demonstrates the paramount importance of population genomics in fungicide discovery.

Cytochrome b gene sequence and structure of Pyrenophora teres and P. tritici-repentis and implications for QoI resistance.

Resistance to QoI fungicides in Pyrenophora teres (Dreschsler) and P. tritici-repentis (Died.) Dreschsler was detected in 2003 in France and in Sweden and Denmark respectively. Molecular analysis revealed the presence of the F129L mutation in resistant isolates of both pathogens. In 2004, the frequency of the F129L mutation in populations of both pathogens further increased. The G143A mutation was also detected in a few isolates of P. tritici-repentis from Denmark and Germany. In 2005, the F129L mutation in P. teres increased in frequency and geographical distribution in France and the UK but remained below 2% in Germany, Switzerland, Belgium and Ireland. In P. tritici-repentis, both mutations were found in a significant proportion of the isolates from Sweden, Denmark and Germany. The G143A mutation conferred a significantly higher level of resistance (higher EC50 values) to Qo inhibitors (QoIs) than did the F129L mutation. In greenhouse trials, resistant isolates with G143A were not well controlled on plants sprayed with recommended field rates, whereas satisfactory control of isolates with F129L was achieved. For the F129L mutation, three different single nucleotide polymorphisms (SNPs), TTA, TTG and CTC, can code for L (leucine) in P. teres, whereas only the CTC codon was detected in P. tritici-repentis isolates. In two out of 250 isolates of P. tritici-repentis from 2005, a mutation at position 137 (G137R) was detected at very low frequency. This mutation conferred similar resistance levels to F129L. The structure of the cytochrome b gene of P. tritici-repentis is significantly different from that of P. teres: an intron directly after amino acid position 143 was detected in P. teres which is not present in P. tritici-repentis. This gene structure suggests that resistance based on the G143A mutation may not occur in P. teres because it is lethal. No G143A isolates were found in any P. teres populations. Although different mutations may evolve in P. tritici-repentis, the G143A mutation will have the strongest impact on field performance of QoI fungicides.