Helge Sierotzki

QoI resistance emerged independently at least 4 times in European populations of Mycosphaerella graminicola

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 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. 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 a west-to-east direction. CONCLUSION 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.

A Review of Current Knowledge of Resistance Aspects for the Next-Generation Succinate Dehydrogenase Inhibitor Fungicides

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.

Fungicide modes of action and resistance in downy mildews

Among oomycetes, Plasmopara viticola on grape and Phytophthora infestans on potato are agronomically the most important pathogens requiring control measures to avoid crop losses. Several chemical classes of fungicides are available with different properties in systemicity, specificity, duration of activity and risk of resistance. The major site-specific fungicides are the Quinone outside inhibitors (QoIs; e.g. azoxystrobin), phenylamides (e.g. mefenoxam), carboxylic acid amides (CAAs; e.g. dimethomorph, mandipropamid) and cyano-acetamide oximes (cymoxanil). In addition, multi-site fungicides such as mancozeb, folpet, chlorothalonil and copper formulations are important for disease control especially in mixtures or in alternation with site-specific fungicides. QoIs inhibit mitochondrial respiration, phenylamides the polymerization of r-RNA, whereas the mode of action of the other two site-specific classes is unknown but not multi-site. The use of site-specific fungicides has in many cases selected for resistant pathogen populations. QoIs are known to follow maternal, largely monogenic inheritance of resistance; they bear a high resistance risk for many but not all oomycetes. For phenylamides, inheritance of resistance is based on nuclear, probably monogenic mechanisms involving one or two semi-dominant genes; resistance risk is high for all oomycetes. The molecular mechanism of resistance to QoIs is mostly based on the G143A mutation in the cytochrome b gene; for phenylamides it is largely unknown. Resistance risk for CAA fungicides is considered as low to moderate depending on the pathogen species. Resistance to CAAs is controlled by two nuclear, recessive genes; the molecular mechanism is unknown. For QoIs and CAAs, resistance in field populations of P. viticola may gradually decline when applications are stopped.

Zymoseptoria tritici: A major threat to wheat production, integrated approaches to control

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.

Paralog Re-Emergence: A Novel, Historically Contingent Mechanism in the Evolution of Antimicrobial Resistance

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.

Resurgence of Pseudoperonospora cubensis: The Causal Agent of Cucurbit Downy Mildew

The downy mildew pathogen, Pseudoperonospora cubensis, which infects plant species in the family Cucurbitaceae, has undergone major changes during the last decade. Disease severity and epidemics are far more destructive than previously reported, and new genotypes, races, pathotypes, and mating types of the pathogen have been discovered in populations from around the globe as a result of the resurgence of the disease. Consequently, disease control through host plant resistance and fungicide applications has become more complex. This resurgence of P. cubensis offers challenges to scientists in many research areas including pathogen biology, epidemiology and dispersal, population structure and population genetics, host preference, host-pathogen interactions and gene expression, genetic host plant resistance, inheritance of host and fungicide resistance, and chemical disease control. This review serves to summarize the current status of this major pathogen and to guide future management and research efforts within this pathosystem.

Isogamous, hermaphroditic inheritance of mitochondrion-encoded resistance to Qo inhibitor fungicides in Blumeria graminis f. sp. tritici.

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).

The cellulose synthase 3 (CesA3) gene of oomycetes: structure, phylogeny and influence on sensitivity to carboxylic acid amide (CAA) fungicides

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.

Resistance mechanism to carboxylic acid amide fungicides in the cucurbit downy mildew pathogen Pseudoperonospora cubensis.

BACKGROUND 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. RESULTS 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. CONCLUSION 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.

Evolution of Qol resistance in Plasmopara viticola oospores

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.