Mutagenesis and Functional Studies with Succinate Dehydrogenase Inhibitors in the Wheat Pathogen Mycosphaerella graminicola

Abstract

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.

Baseline sensitivity and resistance risk assessmemt of Rhizoctonia cerealis to thifluzamide, a succinate dehydrogenase inhibitor

“…Although laboratory and field mutants with resistance to the SDHIs have been reported in eleven plant pathogens, including Ustilago maydis [17], Mycosphaerella graminicola [18][19][20][21][22], Botrytis cinerea [23][24][25][26], Botrytis elliptica from lilies [22], Alternaria alternata from pistachio [27][28][29], Corynespora cassiicola from cucurbits [30,31], Didymella bryoniae from cucurbits [32], Podosphaera xanthii from cucurbits (information from meeting by member of FRAC SDHI working group), Sclerotinia sclerotiorum from oilseed rape [16,21] and Stemphylium botryose from asparagus (information from meeting by member of FRAC SDHI working group), and in phytopathogenic bacteria, including Xanthomanas campestris pv. citri isolates from citrus (Citus reticulata) [33] and Xanthomonas oryzae pv.…”

Mechanism of Action of ME1111, a Novel Antifungal Agent for Topical Treatment of Onychomycosis

“…The enzyme consists of 4 subunits: SdhA, SdhB, SdhC, and SdhD. Agricultural chemicals, such as carboxin and boscalid, exert their antifungal activity by binding to the ubiquinone-binding site of succinate dehydrogenase, which is a cleft formed by amino acid residues from SdhB, SdhC, and SdhD (9)(10)(11). Moreover, several inhibitors that block mitochondrial electron transport have been used in humans as anti-infectives.…”

The origin, versatility and distribution of azole fungicide resistance in the banana black Sigatoka pathogen Pseudocercospora fijiensis

“…This fosters efforts into the research and development for novel chemistry for efficient black Sigatoka control, although alternative products, such as the strobilurins and Succinate Dehydrogenase Inhibitors (SDHIs), are also prone to resistance development (Arango et al 2016;Scalliet et al 2012). Therefore, disease management should embark on the availability of resistant banana germplasms.…”

“…Their fungicidal activity is based on the disruption of the mitochondrial tricarboxylic acid cycle (TCA) through inhibition of the Succinate Dehydrogenase enzyme (SDH). At the molecular level, carboxamides inhibit ubiquinone reduction by binding to the ubiquinone binding site (Qp site) of the SDH enzyme (Scalliet et al 2012).…”

“…At the molecular level, carboxamides inhibit ubiquinone reduction by binding to the ubiquinone binding site (Qp site) of the SDH enzyme (Scalliet et al 2012). New molecules with a wider spectrum of activity were discovered recently (Leroux et al 2010) but are not yet generally applied.…”