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 stoppe

Following the dynamics of strobilurin resistance in Blumeria graminis f.sp. tritici using quantitative allele-specific real-time PCR measurements with the fluorescent dye SYBR Green 1

Strobilurin-resistant isolates of Blumeria (Erysiphe) graminis f.sp. tritici, the cause of wheat powdery mildew, were more than 10-fold less sensitive to azoxystrobin than sensitive isolates. In all resistant isolates, a mutation resulting in the replacement of a glycine by an alanine residue at codon 143 (G143A) in the mitochondrial cytochrome b gene was found. Allele-specific primers were designed to detect this point mutation in infected wheat leaves. Using quantitative fluorescent allele-specific real-time polymerase chain reaction (PCR) measurements, strobilurin-resistant A143 alleles could be detected amongst strobilurin-sensitive G143 alleles at a frequency of at least 1 in 10 000, depending on the amount of target and nontarget DNA. Most isolates tested were dominant homoplasmic for either the A143 or G143 allele, although mixed populations of alleles could be detected in some isolates. In some of these isolates, strobilurin resistance was not always stable when they were maintained for many generations in the absence of selection. The allele-specific real-time PCR assay was also used to follow the dynamics of A143 alleles in field populations of B. graminis f.sp. tritici before and after application of fungicides. As expected, the A143 allele frequency only increased under selection pressure from a strobilurin fungicide. After three sprays of azoxystrobin, a pronounced selection for the strobilurin-resistant allele, with an increase in average frequency from 2.2 to 58 %, was measured. The use of quantitative real-time PCR diagnostics for early detection of fungicide resistance genes at low frequency, coupled with risk evaluation, will be invaluable for further resistance risk assessment and validation of antiresistance strategies

Detection of Zymoseptoria tritici SDHI-insensitive field isolates carrying the SdhC-H152R and SdhD-R47W substitutions

BACKGROUND: Succinate dehydrogenase inhibitor fungicides are important in the management of Zymoseptoria tritici in wheat. New active ingredients from this group of fungicides have been introduced recently and are widely used. Because the fungicides act at a single enzyme site, resistance development in Z. tritici is classified as medium-to-high risk. RESULTS: Isolates from Irish experimental plots in 2015 were tested against the SDHI penthiopyrad during routine monitoring. The median of the population was approximately 2 x less sensitive than the median of the baseline population. Two of the 93 isolates were much less sensitive to penthiopyrad than least sensitive of the baseline isolates. These isolates were also insensitive to most of commercially available SDHIs. Analysis of the succinate dehydrogenase coding genes confirmed the presence of the substitutions SdhC-H152R and SdhD-R47W in the very insensitive isolates. CONCLUSION: This is the first report showing that the SdhC-H152R mutation detected in laboratory mutagenesis studies also exists in the field. The function and relevance of this mutation, combined with SdhD-R47W, still needs to be determined

Simultaneous detection and identification of pathogenic fungi in wheat using a DNA macroarray

The detection of economically important pathogens is a key element in sustainable wheat production and a prerequisite for crop protection. The objective of the project was to develop a DNA macroarray for fast and cost-effective detection of nine pathogenic fungi in wheat: Fusarium graminearum, Fusarium culmorum, Fusarium poae, Microdochium nivale var. majus, Microdochium nivale var. nivale, Puccinia recondita, Septoria tritici, Septoria nodorum and Pyrenophora tritici-repentis. Methodically, a macroarray is similar to a microarray but without the need for expensive equipment. PCR labelled samples of DNA are hybridized to pathogen-specific oligonucleotides (probes) anchored to a solid support. A positive reaction between an amplicon and a perfectly matched oligonucleotide generates a chemiluminescent signal which can be detected by a plate reader. The macroarray is sensitive enough to detect single nucleotide polymorphism (SNPs). Sample analysis is simple, fast, cost-effective, fully automated and suitable for high throughput screening. In this project, the nine wheat pathogens were detected within 6 hours simultaneously in a single sample using between one to four different species-specific probes for each pathogen. Species-specific detector oligonucleotides were designed based on the β-tubulin and/or succinate dehydrogenase region of fungal DNA. The detection limit of the DNA macroarray technique particularly depends on the pathogen-specific oligonucleotides deployed. The necessity for monitoring pathogenic fungi in wheat production and for prediction of crop yield has been recognized for a long time. The DNA macroarray responds very sensitively and has the potential to recognize pathogenic fungi earlier with reference to the cultivation period than a conventional PCR. This means that the DNA macroarray can detect genomic DNA from fungi in a lower potency than the conventional PCR. One benefit of the DNA macroarray for detection of fungal pathogens in wheat is its increased specificity and the other its application to a large number of microorganisms which can be detected in a single assay. This technology has been proven to be relatively cost-effective compared with real-time PCR or microarrays. This project was financially supported by the Commission of Technology and Innovation CTI in Berne, Switzerland

The effect of succinate dehydrogenase inhibitor/azole mixtures on selection of Zymoseptoria tritici isolates with reduced sensitivity

Combining fungicides with different modes of action is regarded as one of the most effective means of slowing the selection of resistance. Field trials were used to study the effects of such mixtures on selection for Zymoseptoria tritici with reduced sensitivity to the succinate dehydrogenase inhibitors (SDHIs) and azole fungicides. The SDHI isopyrazam and the azole epoxiconazole were applied individually as solo products, and together in a preformulated mixture. All fungicide treatments were included at both full and half the recommended doses

Dose-dependent selection drives lineage replacement during the experimental evolution of SDHI fungicide resistance in Zymoseptoria tritici

Fungicide resistance is a constant threat to agricultural production worldwide. Molecular mechanisms of fungicide resistance have been studied extensively in the wheat pathogen Zymoseptoria tritici. However, less is known about the evolutionary processes driving resistance development. In vitro evolutionary studies give the opportunity to investigate this. Here, we examine the adaptation of Z. tritici to fluxapyroxad, a succinate dehydrogenase (Sdh) inhibitor. Replicate populations of Z. tritici derived from the sensitive isolate IPO323 were exposed to increasing concentrations of fluxapyroxad with or without UV mutagenesis. After ten increases in fungicide concentration, sensitivity had decreased dramatically, with replicate populations showing similar phenotypic trajectories. Sequencing the Sdh subunit B, C and D encoding genes identified seven mutations associated with resistance to fluxapyroxad. Mutation frequency over time was measured with a pyrosequencing assay, revealing sequential lineage replacement in the UV mutagenized populations but not in the untreated populations. Repeating selection from set timepoints with different fungicide concentrations revealed that haplotype replacement of Sdh variants was driven by dose-dependent selection as fungicide concentration changed, and was not mutation limited. These findings suggest that fungicide field applications may select for highly insensitive Sdh variants with higher resistance factors if the fungicide concentration is increased to achieve a better disease control. However, in the absence or presence of lower fungicide concentrations, the spread of these strains might be restricted if the underlying Sdh mutations carry fitness penalties

Differentiation in populations of the apple scab fungus Venturia inaequalis on cultivars in a mixed orchard remains over time

The ascomycete Venturia inaequalis causes annual epidemics of apple scab worldwide. Scab development is reduced in mixed cultivar orchards compared with monocultures. To use mixtures in commercial production, we need to understand how the population of scab changes in a mixed orchard and how likely a super race, with virulence factors overcoming multiple resistance factors in the mixed orchard, is to emerge and become dominant. We used short sequence repeat (SSR) markers to investigate the temporal change of scab populations in two mixed cultivar orchards in the UK to infer the likelihood of emergence of a scab super race. There were no significant differences between the populations at the two sampling times (six or seven years apart) in either of the two mixed orchards. In one of the orchards apple scab populations on different cultivars were significantly different and the differences did not diminish over time. These results suggest that it is not inevitable that a super race of V. inaequalis will become dominant during the lifetime of a commercial apple orchard

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

Demethylase Inhibitor Fungicide Resistance in Pyrenophora teres f. sp. teres Associated with Target Site Modification and Inducible Overexpression of Cyp51

Pyrenophora teres f. sp. teres is the cause of net form of net blotch, an economically important foliar disease in barley (Hordeum vulgare). Net and spot forms of net blotch are widely controlled using site-specific systemic fungicides. Although resistance to succinate dehydrogenase inhibitors and quinone outside inhibitors has been addressed before in net blotches, mechanisms controlling demethylation inhibitor resistance have not yet been reported at the molecular level. Here we report the isolation of strains of net form of net blotch in Australia since 2013 resistant to a range of demethylase inhibitor fungicides. Cyp51A:KO103-A1, an allele with the mutation F489L, corresponding to the archetype F495I in Aspergillus fumigatus, was only present in resistant strains and was correlated with resistance factors to various demethylase inhibitors ranging from 1.1 for epoxiconazole to 31.7 for prochloraz. Structural in silico modelling of the sensitive and resistant CYP51A proteins docked with different demethylase inhibitor fungicides showed how the interaction of F489L within the heme cavity produced a localised constriction of the region adjacent to the docking site that is predicted to result in lower binding affinities. Resistant strains also displayed enhanced induced expression of the two Cyp51A paralogs and of Cyp51B genes. While Cyp51B was found to be constitutively expressed in the absence of fungicide, Cyp51A was only detected at extremely low levels. Under fungicide induction, expression of Cyp51B, Cyp51A2 and Cyp51A1 was shown to be 1.6-, 3- and 5.3-fold higher, respectively in the resistant isolate compared to the wild type. These increased levels of expression were not supported by changes in the promoters of any of the three genes. The implications of these findings on demethylase inhibitor activity will require current net blotch management strategies to be reconsidered in order to avoid the development of further resistance and preserve the lifespan of fungicides in use