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ADEPIDYN (pydiflumetofen) is a new broad-spectrum foliar fungicide and the first example of the new group of N-methoxy-(phenethyl)-pyrazole-carboxamides within the class of succinate dehydrogenase inhibitors (SDHIs). It offers effective and long-lasting disease control across multiple crops, protecting them against a broad range of yield- and quality-reducing diseases, including leaf spots, powdery mildew, and molds. In addition, ADEPIDYN delivers as very first SDHI fungicide outstanding performance against Fusarium head blight. It has an excellent crop safety profile whether used on its own, in premixes, or in tank mixes. In addition to its high intrinsic potency, ADEPIDYN is rapidly taken up into the wax layer of the plant leaves, resulting in an excellent quantitative rainfastness. In this chapter, we report the discovery, synthesis, physicochemical, and biokinetic properties as well as biological activity of ADEPIDYN.

BACKGROUND Despite anti-resistance strategies being recommended to reduce selection pressure on insensitive strains, no information is available on fungal population dynamics following their application in real field conditions. In this study, the effects on Plasmopara viticola populations of two identical spray programs, differing only in including or not the carboxylic acid amide (CAA) mandipropamid in mixture and in alternation with an anti-resistance partner, were compared in terms of downy mildew control efficacy and mandipropamid sensitivity in two commercial vineyards for four seasons. RESULTS Both programs effectively and similarly protected grapevine from downy mildew, despite different starting sensitivity levels of the P. viticola populations. In the vineyard where resistant strains were initially present, the frequency of mutations associated with resistance (G1105S/V) fluctuated within seasons in both programs and a shift towards sensitivity occurred after 3 years of the mandipropamid-free program. Where sensitivity was initially present, no changes occurred in the mandipropamid-free program and resistant strains were selected in the mandipropamid program in high disease pressure conditions. CONCLUSION The anti-resistance strategy including mandipropamid in mixture showed a good field performance, but did not completely prevent an increase in the frequency of insensitive strains. This supports the need for appropriate planning to determine which mixtures should be used in the field. © 2018 Society of Chemical Industry.

The Dothideomycete Pseudocercospora fijiensis, previously Mycosphaerella fijiensis, is the causal agent of black Sigatoka, one of the most destructive diseases of bananas and plantains. Disease management depends on fungicide applications, with a major contribution from sterol demethylation-inhibitors (DMIs). The continued use of DMIs places considerable selection pressure on natural P. fijiensis populations, enabling the selection of novel genotypes with reduced sensitivity. The hitherto explanatory mechanism for this reduced sensitivity was the presence of non-synonymous point mutations in the target gene Pfcyp51, encoding the sterol 14α-demethylase enzyme. Here, we demonstrate a second mechanism involved in DMI sensitivity of P. fijiensis. We identified a 19-bp element in the wild-type (wt) Pfcyp51 promoter that concatenates in strains with reduced DMI sensitivity. A polymerase chain reaction (PCR) assay identified up to six Pfcyp51 promoter repeats in four field populations of P. fijiensis in Costa Rica. We used transformation experiments to swap the wt promoter of a sensitive field isolate with a promoter from a strain with reduced DMI sensitivity that comprised multiple insertions. Comparative in vivo phenotyping showed a functional and proportional up-regulation of Pfcyp51, which consequently decreased DMI sensitivity. Our data demonstrate that point mutations in the Pfcyp51 coding domain, as well as promoter inserts, contribute to the reduced DMI sensitivity of P. fijiensis. These results provide new insights into the importance of the appropriate use of DMIs and the need for the discovery of new molecules for black Sigatoka management.

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., Author summary Zymoseptoria tritici is the causal agent of Septoria tritici leaf blotch (STB) of wheat, the most devastating disease for cereal production in Europe. Multiple succinate dehydrogenase inhibitor (SDHI) fungicides have been developed and introduced for the control of STB. We report the discovery and detailed characterization of a paralog of the C subunit of the SDH enzyme conferring standing resistance towards the SHA-SDHIs, a particular chemical subclass of the SDHIs. The SDHC paralog is characterized by its presence/absence, expression and alternative splicing polymorphisms, which in turn influence resistance levels. The identified mechanisms exemplify the importance of population genomics for the discovery and rational design of the most adapted solutions.

Patel, J. S., Meinhardt, S. W., Sierotzki, H., Stammler, G., Gudmestad, N. C., and Adhikari, T. B. 2011. A two-step molecular detection method for Pyrenophora tritici-repentis isolates insensitive to QoI fungicides. Plant Dis. 95:1558-1564. Tan spot, caused by Pyrenophora tritici-repentis, is an important disease of wheat worldwide. To manage tan spot, quinone outside inhibitor (QoI) fungicides such as azoxystrobin and pyraclostrobin have been applied in many countries. QoI fungicides target the cytochrome b (cyt b) site in complex III of mitochondria and, thus, pose a serious risk for resistance development. The resistance mechanism to QoI fungicides is mainly due to point mutations in the cyt b gene. The objective of this study was to develop a molecular detection method for the four currently known mutations responsible for shifts in sensitivity toward QoI fungicides in P. tritici-repentis. Twelve specific primers were designed based on sequences from the National Center for Biotechnology Information accessions AAXI01000704 and DQ919068 and used to generate a fragment of the cyt b gene which possesses four known singlenucleotide polymorphisms (SNPs). These mutant clones served as positive controls because QoI-insensitive and -reduced-sensitive isolates of P. tritici-repentis have not yet been reported in the United States. The partial cyt b gene clones were sequenced to identify the SNPs at sites G143A and F129L. Genomic DNA of the mutated partial cyt b gene clones and the European QoI-insensitive and -reducedsensitive isolates of P. tritici-repentis possessing G143A (GCT) and F129L (TTA, TTG, and CTC) mutations were amplified by polymerase chain reaction (PCR) using two specific primer pairs and were further digested with three specific restriction enzymes (BsaJI, Fnu4HI, and MnlI). The results of the digested PCR product from genomic DNA of known QoI-insensitive and -reduced-sensitive isolates of P. triticirepentis had DNA bands consistent with the mutation GCT at G143A and the mutations TTA, TTG, and CTC at F129L. The amplified region at the F129 site also had 99% sequence similarity with P. teres , the net blotch pathogen of barley. To validate mutations, we further tested two isolates of P. teres known to have reduced sensitivity to QoI fungicides possessing the mutations TTA and CTC at F129L. After PCR amplification and restriction digestion, DNA bands identical to those observed for the partial cyt b mutant clones were detected. These results suggest that this newly developed two-step molecular detection method is rapid, robust, and specific to monitor QoI-insensitive and -reducedsensitive isolates of P. tritici-repentis.

Syngenta is one of the major agrochemical companies with enormous breadth of technologies in Crop Protection, Seeds and Seed Care. Through an exceptionally broad product range and research investment, we are not only able to provide the grower with integrated offers now but also truly innovative and transformative technologies in the future. In this commentary Syngenta scientists give their views on the key wheat pathogen Zymoseptoria tritici from its business importance in Europe, the way we screen new Z. tritici fungicides, the way we monitor the evolution of fungicide resistance and breed for Z. tritici resistance. These four points are continuously revisited and adapted during the development of new fungicides, and academic collaborations are critically important to stay at the fore front of developments in cell biology, physiology and genetic research.

Frogeye leaf spot of soybean, caused by the fungus Cercospora sojina, reduces soybean yields in most of the top-producing countries around the world. Control strategies for frogeye leaf spot can rely heavily on quinone outside inhibitor (QoI) fungicides. In 2010, QoI fungicide-resistant C. sojina isolates were identified in Tennessee for the first time. As the target of QoI fungicides, the cytochrome b gene present in fungal mitochondria has played a key role in the development of resistance to this fungicide class. The cytochrome b genes from three QoI-sensitive and three QoI-resistant C. sojina isolates were cloned and sequenced. The complete coding sequence of the cytochrome b gene was identified and found to encode 396 amino acids. The QoI-resistant C. sojina isolates contained the G143A mutation in the cytochrome b gene, a guanidine to cytosine transversion at the second position in codon 143 that causes an amino acid substitution of alanine for glycine. C. sojina-specific polymerase chain reaction primer sets and TaqMan probes were developed to efficiently discriminate QoI-resistant and -sensitive isolates. The molecular basis of QoI fungicide resistance in field isolates of C. sojina was identified as the G143A mutation, and specific molecular approaches were developed to discriminate and to track QoI-resistant and -sensitive isolates of C. sojina.

Despite anti-resistance strategies being recommended to reduce selection pressure on insensitive strains, no information is available on fungal population dynamics following their application in real field conditions. In this study, the effects on Plasmopara viticola populations of two identical spray programs, differing only in including or not the carboxylic acid amide (CAA) mandipropamid in mixture and in alternation with an anti-resistance partner, were compared in terms of downy mildew control efficacy and mandipropamid sensitivity in two commercial vineyards for four seasons.Both programs effectively and similarly protected grapevine from downy mildew, despite different starting sensitivity levels of the P. viticola populations. In the vineyard where resistant strains were initially present, the frequency of mutations associated with resistance (G1105S/V) fluctuated within seasons in both programs and a shift towards sensitivity occurred after 3 years of the mandipropamid-free program. Where sensitivity was initially present, no changes occurred in the mandipropamid-free program and resistant strains were selected in the mandipropamid program in high disease pressure conditions.The anti-resistance strategy including mandipropamid in mixture showed a good field performance, but did not completely prevent an increase in the frequency of insensitive strains. This supports the need for appropriate planning to determine which mixtures should be used in the field. © 2018 Society of Chemical Industry.

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.

Proper disease control is very important to minimize yield losses caused by oomycetes in many crops. Today, oomycete control is partially achieved by breeding for resistance, but mainly by application of single-site mode of action fungicides including the carboxylic acid amides (CAAs). Despite having mostly specific targets, fungicidal activity can differ even in species belonging to the same phylum but the underlying mechanisms are often poorly understood. In an attempt to elucidate the phylogenetic basis and underlying molecular mechanism of sensitivity and tolerance to CAAs, the cellulose synthase 3 (CesA3) gene was isolated and characterized, encoding the target site of this fungicide class. The CesA3 gene was present in all 25 species included in this study representing the orders Albuginales, Leptomitales, Peronosporales, Pythiales, Rhipidiales and Saprolegniales, and based on phylogenetic analyses, enabled good resolution of all the different taxonomic orders. Sensitivity assays using the CAA fungicide mandipropamid (MPD) demonstrated that only species belonging to the Peronosporales were inhibited by the fungicide. Molecular data provided evidence, that the observed difference in sensitivity to CAAs between Peronosporales and CAA tolerant species is most likely caused by an inherent amino acid configuration at position 1109 in CesA3 possibly affecting fungicide binding. The present study not only succeeded in linking CAA sensitivity of various oomycetes to the inherent CesA3 target site configuration, but could also relate it to the broader phylogenetic context.

Wild type (WT) field isolates of Bremia lactucae failed to germinate in vitro or infect lettuce leaves in the presence of CAA (carboxylic acid amide) fungicides. Minimal inhibitory concentrations (MIC) for mandipropamid, dimethomorph, benthiavalicarb and iprovalicarb were 0.005, 0.5, 0.5 and 5 μg ml−1, respectively. Mutagenesis experiments showed that spores exposed to EMS (ethyl methane sulphonate) or UV irradiation (254 nm) could infect lettuce leaves in the presence of up to 100 μg ml−1 CAA. The proportion of infected leaves relative to the number of spores inoculated (infection frequency) was inversely related to the concentration of CAA used, ranging between 0 and 160 per 1 × 106 spores. Resistant mutants (RM) lost their resistance within 1–14 reproduction cycles on CAA-treated plants. Crosses were made between RMxWT isolates and RMxRM isolates with an attempt to obtain stable homozygous resistant off-springs. Such crosses yielded few resistant but unstable progeny isolates. Mutagenic treatments given to hybrid isolates also failed to produce stable resistance. Previous gene sequencing data showed that stable resistance to CAAs is based on a single SNP in the cellulose synthase 3 (CesA3) gene of Plasmopara viticola. Therefore, we sequenced a 582 bp DNA fragment of Ces3A of WT, RM and hybrid isolates of B.lactucae. No mutation in this gene fragment was found. We conclude that mutagenic agents like EMS or UV may induce resistance to CAA in Bremia lactucae but this resistance is not stable and not linked to mutations in CesA3 gene.

A total of 241 isolates of Phytophthora infestans were collected in 1997, 2006 and 2007 in eight European countries and characterized with molecular markers (simple sequence repeats, SSR genotypes) and phenotypic traits such as sensitivity to fungicides, mating type and aggressiveness. The mating type distribution changed from mainly A1 in 1997 to a majority of A2 in 2007. No resistant isolates were detected for fluazinam and mandipropamid, whereas the proportion of isolates resistant to mefenoxam (MFX) was high and increased over the years. There was no genetic link between mating type and MFX resistance. Aggressiveness (product between lesion expansion and sporulation capacity) was slightly higher for MFX-resistant compared to sensitive isolates and for isolates collected later compared to earlier in the same season. It was about equally high for A1 and A2 types, and for French isolates in 1997 and British isolates in 2007, but lower for French isolates in 2007. Six different SSR genotype families were distinguished. In 1997, populations were dominated by genotype families I and III/IV, which significantly declined in 2007 being largely displaced by genotype families II ('blue 13' type) and V, which are by coincidence mainly A2 MFX resistant and A1 MFX sensitive, respectively. However, mating type and MFX resistance were genetically not linked to SSR genotypes.

QoI resistance in P. viticola was first detected in France and Italy in 1999. Molecular and biological assays have been carried out since 2000 in order to provide reliable methods of detecting and quantifying resistance. Oospores were collected in vineyards located in northern and southern Italy. QoI resistance was evaluated by the germination rate of oospores on azoxystrobin amended medium and the frequency of mutant alleles in the DNA extracted from oospores. Both methods correlated to each other and were used side by side to test QoI resistance. Due to the spontaneous occurrence of the G143A mutation in wild type populations and the immigration from surrounding vineyards, resistance frequencies up to 10% were found in samples collected from vineyards never treated with QoIs. Particularly high values, about 90%, were associated with the application of five to six QoI treatments within the same season, while lower percentages, about 30%, were detected in vineyards treated with QoI used in mixture with fungicides belonging to a different resistance group. A progressive decrease of resistance frequency was observed when QoI applications were reduced in number or completely suspended for at least one season. Therefore, a full recovery of sensitivity may be achieved even in vineyards characterized by high levels of resistance, if particular care is taken during disease control by using QoIs only in mixtures and reducing the number of QoI treatments.

BACKGROUND: QoI fungicides or quinone outside inhibitors (also called strobilurins) have been widely used to control agriculturally important fungal pathogens since their introduction in 1996. Strobilurins block the respiration pathway by inhibiting the cytochrome bc1 complex in mitochondria. Several plant pathogenic fungi have developed field resistance. The firstQoIresistanceinMycosphaerellagraminicola(Fuckel)SchroterwasdetectedretrospectivelyinUKin2001atalowfrequency inQoI-treatedplots. Duringthefollowing seasons,resistancereachedhighfrequenciesacross northernEurope. Theaimof this study was to identify the main evolutionary forces driving the rapid emergence and spread of QoI resistance in M. graminicola populations. RESULTS: The G143A mutation causing QoI resistance was first detected during 2002 in all tested populations and in eight distinct mtDNA sequence haplotypes. By 2004, 24 different mtDNA haplotypes contained the G143A mutation. Phylogenetic analysis showed that strobilurin resistance was acquired independently through at least four recurrent mutations at the same site of cytochrome b. Estimates of directional migration rates showed that the majority of gene flow in Europe had occurred in aw est-to-east direction. CONCLUSION: This study demonstrated that recurring mutation si ndependently introduced the QoI resistance allele into different genetic and geographic backgrounds. The resistan th aplotypes then increased in frequency owing to the strong fungicide selection and spread eastward through wind dispersal of ascospores. c ! 2008 Society of Chemical Industry

Sensitivity to the new carboxylic acid amide (CAA) fungicide mandipropamid (MPD) in Phytophthora infestans was measured for isolates collected between 1989 and 2002 in Israel prior to the commercial use of MPD (baseline sensitivity, 44 isolates), and from MPD-treated (25 isolates) and untreated fields (215 isolates) in nine European countries and Israel between 2001 and 2005. All isolates were sensitive to MPD, with EC50 values ranging between 0·02 and 2·98 µg mL−1. Plastic-tunnel (UK), shade-house (Israel) and field experiments (Israel) conducted during 2001–05 showed that enforced selection pressure, applied preventively or curatively, imposed by repeated sublethal (5 µg mL−1) or excessive (500–1000 µg mL−1) doses of MPD on mixed isolates of P. infestans produced no isolates resistant to the compound. The results of this study indicate that the probability of a buildup of resistant sub-populations of P. infestans to mandipropamid in the field is low.

Mandipropamid is a new mandelic acid amide fungicide expressing high activity against foliar infecting oomycetes, including the grapevine downy mildew, Plasmopara viticola. Because cross-resistance with the valinamide fungicides iprovalicarb and benthiavalicarb and the cinnamic acid amide fungicides dimethomorph and flumorph was postulated, all five compounds are classified as carboxylic acid amide (CAA) fungicides. To support this classification, cross-resistance among these compounds with field isolates and the segregation of resistance in F1 and F2 progeny of P. viticola were evaluated. A bimodal distribution of sensitivity in field isolates and cross-resistance among all CAAs for the vast majority of isolates were detected. Crosses between sensitive (s) and CAA-resistant (r) isolates of opposite mating types, P1 and P2, yielded abundant oospores. All F1-progeny isolates were sensitive to CAAs (s:r segregation 1:0), whereas in F2 progeny segregation of about 9:1 (s:r) was observed suggesting that resistance to CAA fungicides is controlled by two recessive nuclear genes. Mating type segregated in a ratio P1:P2 of c. 2:1 in F1 and 1:1 in F2 progeny. In the same crosses, resistance to the phenylamide fungicide mefenoxam segregated in a ratio of c. 1:3:2 (sensitive:intermediate:resistant), reflecting the monogenic, semidominant nature of resistance. The risk of resistance in P. viticola was classified as high for phenylamide and moderate for CAA fungicides. This is the first report on the inheritance of phenotypic traits in P. viticola.

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.

QoI fungicides, inhibitors of mitochondrial respiration at the Qo site of cytochrome b in the mitochondrial bc1 enzyme complex, are commonly applied in vineyards against Plasmopara viticola (Berk. & MA Curtis) Berl. & De Toni. Numerous treatments per year with QoI fungicides can lead to the selection of resistant strains in the pathogen population owing to the very specific and efficient mode of action. In order to evaluate the resistance risk and its development, two different methods, biological and molecular, were applied to measure the sensitivity of oospores differentiated in vineyards, both treated and untreated with azoxystrobin, from 2000 to 2004. Assays using oospores have the advantage of analysing the sensitivity of bulked samples randomly collected in vineyards, describing accurately the status of resistance at the end of the grapevine growing season. Both methods correlated well in describing the resistance situation in vineyards. QoI resistance was not observed in one vineyard never treated with QoI fungicides. In the vineyard where azoxystrobin had been used in mixture with folpet, the selection of QoI-resistant strains was lower, compared with using solely QoI. In vineyards where QoI treatments have been stopped, a decrease in resistance was generally observed. Copyright © 2006 Society of Chemical Industry

Zymoseptoria tritici is a dominant pathogen in wheat causing Septoria leaf blotch (SLB), and sterol 14α-demethylation inhibitors fungicides (DMI) are commonly used for control in Northern Europe. In 14 winter wheat trials carried out in Denmark, Lithuania, and Sweden in the years 2011 to 2013, fungicides containing DMIs were investigated for their efficacy and impact on CYP51 mutations in Z. tritici populations. All fungicide treatments were applied twice – each time using 50 % of the standard rate, applied at GS 37 & 55. The single-agent DMIs, epoxiconazole and prothioconazole and a mixture of difenoconazole + propiconazole gave similar control and crop yields. The best solution varied among localities. Adding prochloraz to prothioconazole as well as using co-formulations of DMIs + SDHIs generally improved control compared with using DMIs alone. Yield responses were significant from all treatments, but co-formulations of DMIs plus SDHIs increased yield the most. Specific CYP51 mutations in Z. tritici were analysed by pyrosequencing and qPCR. Their frequency varied across sites and countries. The amount of I381V was high in all trials whereas the amount of A379G was moderate. Levels of D134G, V136C, and S524T were low to moderate across all sites. DMIs and mixtures of DMIs + SDHI selected differently for CYP51 mutations. Prochloraz increased selection for D134G and V136A and decreased selection for A379G and I381V. The lowest selection pressure towards D134G and V136A/C was recorded in the presence of the mixture difenoconazole and propioconazole. Mixtures of DMIs and SDHI tended to lower the frequency of V136A/C compared to DMIs used alone but had no measurable impact on the frequency of I381V and A379G. No responses were seen in relation to S524T, which only occurred at a very low level.

Respiration inhibitor fungicides are highly successful in controlling a wide range of plant pathogens. The biggest group is the QoI fungicides possessing activity toward many major pathogens within Deuteromycetes, Ascomycetes, Basidiomycetes, and Oomycetes. The inhibitor binding to the target pocket is well investigated, and also the resistance mechanism is exhaustively characterized. Resistance to QoIs is based on a point mutation, G143A, as main mechanism; also F129L and G137R can cause resistance but with minor relevance. Resistance is widespread in many plant pathogens and evolved rather quickly in high-risk pathogen/host systems, like powdery mildew on cereals or downy mildew on grapes. In pathogens considered as low risk for resistance development, resistance has developed less rapidly and only recently after more than 10 years of usage, such as in Rhizoctonia solani on rice. In rust pathogens, resistance to QoIs has not developed until today based on the presence of a bi2 intron after position 143 in the cyt b gene. The QiI and the recently launched QoSI fungicides are active against oomycete pathogens. QiIs bind to a different, inner heme pocket of the cytochrome bc1 complex. Confirmed field resistance has been observed so far only in Phytophthora capsici; it is based on an unknown mutation. QoSIs bind to the distal area of the Qo heme pocket of the cytochrome b complex and are not cross-resistant to QoI fungicides. Resistance has not yet been observed in field populations. Respiration inhibitors are still one of the most frequently used fungicide groups for controlling plant diseases. Their success in the market is based on the spectrum of activity, the beneficial effects on plant physiology, and also the strict and well-accepted use recommendations.

Oomycetes are “fungal-like” protista that are phylogenetically distinct from fungi, they are diploid, and cell walls contain cellulose. Oomycetes comprise major plant pathogens within the orders Saprolegniales, Pythiales, Peronosporales (“downy mildews” including species of Plasmopara, Phytophthora, Peronospora, Pseudoperonospora, Bremia), and Sclerosporales. Control of oomycete diseases relies mainly on chemical measures using products within 16 different chemical groups, among which the phenylamides (PAs), quinone outside inhibitors (QoIs), carboxylic acid amides (CAAs), and multisite inhibitors are most widely used. However, resistance evolved against most single-site inhibitors in many oomycete pathogen species. Resistance against PAs (inhibitors of RNA polymerase I) was detected in most oomycete pathogen species; the molecular mechanism was associated recently with the presence of the Y382F mutation in the RNApolI gene. QoIs inhibit electron transport in complex III of the respiration chain; two major mutations, G143A (in Plasmopara viticola) and F129L (in Pythium spp.), have been identified in the cytochrome b, cyt b gene coding for resistance. However, no resistant isolates were observed in Phytophthora species. CAAs inhibit cellulose synthase; several mutations, G1105S/V (in P. viticola) and G1105V/W (in Pseudoperonospora cubensis), were detected in the cellulose synthase, CesA3 gene of CAA-resistant field isolates. In Phytophthora infestans and P. capsici, no CAA-resistant field isolates were discovered, whereas in artificial mutants, the mutations G1105A/V and V1109L/M were found. The L1109 and M1109 amino acid configurations are responsible for the intrinsic insensitivity to CAAs of the entire genus Pythium and all oomycetes outside Peronosporales. Resistance is inherited by one recessive nuclear gene (CesA3) for CAAs, one maternal mitochondrial gene (cytb) for QoIs, and mainly one semidominant nuclear gene for PAs. The consequences of the molecular mechanisms for evolution and stability of resistance are discussed in the light of oomycete pathogen biology.

A fragment of the mitochondrial cytochrome b (cyt b) gene of 13 agronomically important plant pathogenic Basidiomycetes was sequenced, including several Puccinia spp., Uromyces appendiculatus, Phakopsora pachyrhizi, Hemileia vastatrix and Rhizoctonia solani. The deduced amino acid sequences (residues 142–266) were used to study the relatedness of these pathogens as compared to other species of the Basidiomycetes, Ascomycetes and Oomycetes. The relatedness was also studied at nuclear level using the Internal Transcribed Spacers (ITS) in the ribosomal DNA. Phylogenic trees were constructed with the maximum parsimony (MP) and the neighbour-joining (NJ) methods. On the basis of both cytochrome b and ITS sequences, the Puccinia species pathogenic to graminaceous crop plants, such as Puccinia recondita f. sp. tritici, Puccinia graminis f. sp. tritici, Puccinia striiformis f. sp. tritici, Puccinia coronata f. sp. avenae, Puccinia hordei, P. recondita f. sp. secalis and Puccinia sorghi, together with Puccinia horiana from Chrysanthemum, were very closely related to each other, whereas Puccinia arachidis (from peanut) was closely related to U. appendiculatus (from beans) but more distant from the other Puccinia species. Both rusts on soybean (P. pachyrhizi) and coffee (H. vastatrix) were outside the Puccinia cluster. All rusts were separated from other Basidiomycetes such as R. solani and the strobilurin producing species Strobilurus tenacellus and Mycena galopoda. Our results demonstrate that the amino acid sequence of the mitochondrial cytochrome b is a valid tool to study phylogenic relatedness among plant pathogenic Basidiomycetes and supports taxonomic grouping based on morphological structures and host specificity. Because of their high variability, ITS sequences were able to discriminate Puccinia species, which were identical on the basis of the cytochrome b amino acid sequence. Thus, ITS sequences could better show differences among species or within a species, whereas cytochrome b is more suitable than ITS for phyologenic inference at family or genus level. In addition, the sequence data obtained during this study represent essential information for easy isolation of the cyt b gene and detection of point mutations conferring resistance to Qo Inhibitor fungicides that eventually may evolve.

The fragment of the cytochrome b (cyt b) gene responsible for the binding site of QoI fungicides was sequenced for different Puccinia species by using DNA and RNA as template for PCR and RT-PCR, respectively. Degenerated primers for the cyt b gene amplified in P. recondita f.sp. tritici a 450 bp fragment, which was cloned and sequenced. At cDNA level, several Thermal Asymmetric InterLaced (TAIL)-PCR cycles were needed to produce a 996 bp long fragment, which corresponded to almost the whole cyt b gene (about 1160–1180 bp, without introns). This fragment was sequenced and specific primers were designed. Amplification with cyt b specific primers using genomic DNA as template revealed the presence of an intron of about 1500 bp length after the codon for glycine at amino acid position 143. By using the same primer pair, the cyt b gene fragment was amplified and sequenced both at cDNA and genomic DNA level also for other rust species, including P. graminis f.sp. tritici (length: 506 bp), P. striiformis f.sp. tritici (755 bp), P. coronata f.sp. avenae (644 bp), P. hordei (660 bp), P. recondita f.sp. secalis (687 bp), P. sorghi (709 bp), and P. horiana (478 bp). At the same position as for P. recondita f.sp. tritici, an intron of about 1500–1600 bp length was detected also in all other Puccinia species. High homologies were observed among all Puccinia species for both the exonic and intronic fragments of the cyt b gene. Specific primers for the cyt b gene of all eight Puccinia species were developed, which easily amplified the fragment of the gene including all possible mutations known to confer resistance to QoIs in several plant pathogens. However, in all tested isolates of the Puccinia species included in this study, the sequence of cyt b gene fragment did not contain any point mutations.

A single nucleotide polymorphism (snp) in the cytochrome b gene confers resistance to strobilurin fungicides in Erysiphe graminis DC f sp tritici Marchal. On the basis of this point mutation three different types of molecular markers have been developed. Cleaved amplified polymorphic sequences and allele-specific PCR were used to score resistant and sensitive isolates from specifically selected regional populations across Europe. The results of molecular tests were in total agreement with the resistance phenotypes revealed by in vivo tests. Serial dilutions of mixed samples (resistant/sensitive) delimited the detection for strobilurin-resistant alleles to a range of 10–50% for both marker classes. Due to these detection limits no mixture of mitochondria within individual isolates was found. Denaturing high performance chromatography was used to increase the detection sensitivity for the mutant allele. Although the detection limit was lowered to 5–10%, there was no evidence for the existence of mixed mitochondrial genotypes. © 2003 Society of Chemical Industry

Evolution of resistance to drugs and pesticides poses a serious threat to human health and agricultural production. CYP51 encodes the target site of azole fungicides, widely used clinically and in agriculture. Azole resistance can evolve due to point mutations or overexpression of CYP51, and previous studies have shown that fungicide-resistant alleles have arisen by de novo mutation. Paralogs CYP51A and CYP51B are found in filamentous ascomycetes, but CYP51A has been lost from multiple lineages. Here, we show that in the barley pathogen Rhynchosporium commune, re-emergence of CYP51A constitutes a novel mechanism for the evolution of resistance to azoles. Pyrosequencing analysis of historical barley leaf samples from a unique long-term experiment from 1892 to 2008 indicates that the majority of the R. commune population lacked CYP51A until 1985, after which the frequency of CYP51A rapidly increased. Functional analysis demonstrates that CYP51A retains the same substrate as CYP51B, but with different transcriptional regulation. Phylogenetic analyses show that the origin of CYP51A far predates azole use, and newly sequenced Rhynchosporium genomes show CYP51A persisting in the R. commune lineage rather than being regained by horizontal gene transfer; therefore, CYP51A re-emergence provides an example of adaptation to novel compounds by selection from standing genetic variation.

The sensitivity of Venturia inaequalis field isolates to inhibitors of the cytochrome bc1 complex at the Qo site (QoIs) was characterised at the molecular, biochemical and physiological level, and compared to other respiration inhibitors. Comparison of a sensitive and a QoI-resistant isolate revealed very high resistance factors both in mycelium growth and spore germination assays. Cross-resistance was observed among QoIs such as trifloxystrobin, azoxystrobin, famoxadone, strobilurin B and myxothiazol. In the mycelium growth assay, antimycin A, an inhibitor of the cytochrome bc1 complex at the Qi site, was less active against the QoI-resistant than against the sensitive isolate. The mixture of QoIs with salicylhydroxamic acid (SHAM), an inhibitor of the alternative oxidase, exerted synergistic effects in the spore germination but not in the mycelium growth assay. Thus, the cytochrome and the alternative respiration pathways are assumed to play different roles, depending on the developmental stage of the fungus. Induction of alternative oxidase (AOX) by trifloxystrobin was observed in mycelium cells at the molecular level for the sensitive but not the resistant isolate. Following QoI treatment, respiration parameters such as oxygen consumption, ATP level, membrane potential and succinate dehydrogenase activity were only slightly reduced in Qo-resistant mycelium cells, and remained at much higher levels than in sensitive cells. In contrast, no difference was observed between sensitive and resistant isolates when NADH consumption was measured. Comparison of the cytochrome b (cyt b) gene of the sensitive and resistant isolates did not reveal any point mutations as is known to occur in resistant isolates of other plant pathogens. It is assumed that QoI resistance in V inaequalis may be based on a compensation of the energy deficiency following QoI application upstream of the NADH dehydrogenase of the respiratory chain. © 2001 Society of Chemical Industry

Field isolates of Mycosphaerella fijiensis, causing black Sigatoka of banana, were characterised for their sensitivity to different inhibitors of the cytochrome bc1 enzyme complex (Qo respiration inhibitors, strobilurin fungicides), using physiological, biochemical and molecular genetic methods. Strobilurin-resistant isolates exhibited very high resistance factors both in mycelial growth inhibition and NADH consumption assays. Cross-resistance was observed among all Qo inhibitors, including trifloxystrobin, azoxystrobin, famoxadone, strobilurin B and myxothiazol. However, the Qi and the cytochrome aa3 inhibitors, antimycin A and potassium cyanide, respectively, were not cross-resistant to Qo inhibitors. In sensitive but not in resistant isolates, mixtures of Qo inhibitors and SHAM, an inhibitor of the alternative oxidase (AOX), were more active than the components alone, indicating that the alternative pathway is essential in metabolism, but not causal for resistance. In the cell-free NADH-consumption assay, the Qo inhibitors affected the sensitive but not the resistant isolates, suggesting that AOX was not active in sub-mitochondrial particles. In whole cells, however, the AOX has a basic expression level and is probably not inducible by trifloxystrobin. Sequencing of the cytochrome b gene of sensitive and resistant M fijiensis isolates revealed a difference in the nucleotide sequence leading to a single amino acid change from glycine to alanine at position 143 in the resistant isolate. This change is known to occur also in the naturally tolerant basidiomycete Mycena galopoda. It is suggested that the field isolates of M fijiensis can acquire resistance to Qo inhibitors due to a target site alteration with a single base pair change. Resistant isolates do not seem to contain a mixture of mutated and non-mutated DNA, indicating a complete selection of resistant mitochondria and a maternally donated mode of resistance. © 2000 Society of Chemical Industry

Pseudoperonospora cubensis, the causal oomycete agent of cucurbit downy mildew, is responsible for enormous crop losses in many species of Cucurbitaceae, particularly in cucumber and melon. Disease control is mainly achieved by combinations of host resistance and fungicide applications. However, since 2004, resistance to downy mildew in cucumber has been overcome by the pathogen, thus driving farmers to rely only on fungicide spray applications, including carboxylic acid amide (CAA) fungicides. Recently, CAA-resistant isolates of P. cubensis were recovered, but the underlying mechanism of resistance was not revealed. The purpose of the present study was to identify the molecular mechanism controlling resistance to CAAs in P. cubensis.The four CesA (cellulose synthase) genes responsible for cellulose biosynthesis in P. cubensis were characterised. Resistant strains showed a mutation in the CesA3 gene, at position 1105, leading to an amino acid exchange from glycine to valine or tryptophan. Cross-resistance tests with different CAAs indicated that these mutations lead to resistance against all tested CAAs.Point mutations in the CesA3 gene of P. cubensis lead to CAA resistance. Accurate monitoring of these mutations among P. cubensis populations may improve/facilitate adequate recommendation/deployment of fungicides in the field.

QoI fungicides or quinone outside inhibitors (also called strobilurins) have been widely used to control agriculturally important fungal pathogens since their introduction in 1996. Strobilurins block the respiration pathway by inhibiting the cytochrome bc1 complex in mitochondria. Several plant pathogenic fungi have developed field resistance. The first QoI resistance in Mycosphaerella graminicola (Fuckel) Schroter was detected retrospectively in UK in 2001 at a low frequency in QoI-treated plots. During the following seasons, resistance reached high frequencies across northern Europe. The aim of this study was to identify the main evolutionary forces driving the rapid emergence and spread of QoI resistance in M. graminicola populations.The G143A mutation causing QoI resistance was first detected during 2002 in all tested populations and in eight distinct mtDNA sequence haplotypes. By 2004, 24 different mtDNA haplotypes contained the G143A mutation. Phylogenetic analysis showed that strobilurin resistance was acquired independently through at least four recurrent mutations at the same site of cytochrome b. Estimates of directional migration rates showed that the majority of gene flow in Europe had occurred in a west-to-east direction.This study demonstrated that recurring mutations independently introduced the QoI resistance allele into different genetic and geographic backgrounds. The resistant haplotypes then increased in frequency owing to the strong fungicide selection and spread eastward through wind dispersal of ascospores.

Of 20 Plasmopara viticola isolates collected in four locations in Virginia and northwest North Carolina in 2005, 16 were resistant to QoI fungicides. The resistance factor was over 100, and label rates of formulated azoxystrobin and pyraclostrobin provided little or no control of these isolates. Additional sampling in 2006 revealed at least 15 additional vineyards with QoI-resistant P. viticola in Virginia, Maryland, and Pennsylvania. Of 22 isolates of Erysiphe (Uncinula) necator collected in 2005 from five Virginia locations, 20 isolates from 4 locations showed resistance to QoI fungicides. The G143A mutation for resistance was detected in several isolates of both pathogens. This is the first detection of this type of resistance in P. viticola in North America, and the second North American report of QoI resistance in E. necator. Accepted for publication 26 November 2007. Published 11 February 2008.

The cytochrome b (cyt b) gene structure was characterized for different agronomically important plant pathogens, such as Puccinia recondita f sp tritici (Erikss) CO Johnston, P graminis f sp tritici Erikss and Hennings, P striiformis f sp tritici Erikss, P coronata f sp avenae P Syd & Syd, P hordei GH Otth, P recondita f sp secalis Roberge, P sorghi Schwein, P horiana Henn, Uromyces appendiculatus (Pers) Unger, Phakopsora pachyrhizi Syd & P Syd, Hemileia vastatrix Berk & Broome, Alternaria solani Sorauer, A alternata (Fr) Keissl and Plasmopara viticola (Berk & Curt) Berlese & de Toni. The sequenced fragment included the two hot spot regions in which mutations conferring resistance to QoI fungicides may occur. The cyt b gene structure of these pathogens was compared with that of other species from public databases, including the strobilurin-producing fungus Mycena galopoda (Pers) P Kumm, Saccharomyces cerevisiae Meyer ex Hansen, Venturia inaequalis (Cooke) Winter and Mycosphaerella fijiensis Morelet. In all rust species, as well as in A solani, resistance to QoI fungicides caused by the mutation G143A has never been reported. A type I intron was observed directly after the codon for glycine at position 143 in these species. This intron was absent in pathogens such as A alternata, Blumeria graminis (DC) Speer, Pyricularia grisea Sacc, Mycosphaerella graminicola (Fuckel) J Schrot, M fijiensis, V inaequalis and P viticola, in which resistance to QoI fungicides has occurred and the glycine is replaced by alanine at position 143 in the resistant genotype. The present authors predict that a nucleotide substitution in codon 143 would prevent splicing of the intron, leading to a deficient cytochrome b, which is lethal. As a consequence, the evolution of resistance to QoI fungicides based on G143A is not likely to evolve in pathogens carrying an intron directly after this codon.

A single nucleotide polymorphism (snp) in the cytochrome b gene confers resistance to strobilurin fungicides in Erysiphe graminis DC fsp tritici Marchal. On the basis of this point mutation three different types of molecular markers have been developed. Cleaved amplified polymorphic sequences and allele-specific PCR were used to score resistant and sensitive isolates from specifically selected regional populations across Europe. The results of molecular tests were in total agreement with the resistance phenotypes revealed by in vivo tests. Serial dilutions of mixed samples (resistant/sensitive) delimited the detection for strobilurin-resistant alleles to a range of 10-50% for both marker classes. Due to these detection limits no mixture of mitochondria within individual isolates was found. Denaturing high performance chromatography was used to increase the detection sensitivity for the mutant allele. Although the detection limit was lowered to 5-10%, there was no evidence for the existence of mixed mitochondrial genotypes.

A mutation of glycine to alanine at position 143 in the mitochondrial cytochrome b amino acid sequence of Blumeria graminis f. sp. tritici cosegregated with the QoI-resistant phenotype in a ratio of 1:1 in a cross between a sensitive and a resistant isolate. This mutation was used as a mitochondrial marker to determine whether mitochondrial inheritance in B. graminis was anisogamous, as in heterothallic Neurospora sp., or isogamous and hermaphroditic, as in Aspergillus nidulans. Segregation of mitochondrial genotypes in B. graminis f. sp. tritici was consistent with inheritance of mitochondria being hermaphroditic and isogamous, in that all ascospores from an individual cleistothecium had the same mitochondrial genotype and that either parent could act as the maternal parent of a cleistothecium. Within each cleistothecium, nuclear segregation occurred independently of mitochondrial inheritance, as shown by segregation of resistance to the fungicide triadimenol and by segregation of avirulences to the wheat cultivars Galahad (Pm2), Armada (Pm4b), and Holger (Pm6).