Predicting Resistance by Mutagenesis: Lessons from 45 Years of MBC Resistance

When a new fungicide class is introduced, it is useful to anticipate the resistance risk in advance, attempting to predict both risk level and potential mechanisms. One tool for the prediction of resistance risk is laboratory selection for resistance, with the mutational supply increased through UV or chemical mutagenesis. This enables resistance to emerge more rapidly than in the field, but may produce mutations that would not emerge under field conditions.The methyl-benzimidazole carbamates (MBCs) were the first systemic single-site agricultural fungicides, and the first fungicides affected by rapid evolution of target-site resistance. MBC resistance has now been reported in over 90 plant pathogens in the field, and laboratory mutants have been studied in nearly 30 species.The most common field mutations, including β-tubulin E198A/K/G, F200Y and L240F, have all been identified in laboratory mutants. However, of 28 mutations identified in laboratory mutants, only nine have been reported in the field. Therefore, the predictive value of mutagenesis studies would be increased by understanding which mutations are likely to emerge in the field.Our review of the literature indicates that mutations with high resistance factors, and those found in multiple species, are more likely to be reported in the field. However, there are many exceptions, possibly due to fitness penalties. Whether a mutation occurred in the same species appears less relevant, perhaps because β-tubulin is highly conserved so functional constraints are similar across all species. Predictability of mutations in other target sites will depend on the level and conservation of constraints

Azole sensitivity in Leptosphaeria pathogens of oilseed rape: the role of lanosterol 14α-demethylase

Lanosterol 14-α demethylase is a key enzyme intermediating the biosynthesis of ergosterol in fungi, and the target of azole fungicides. Studies have suggested that Leptosphaeria maculans and L. biglobosa, the causal agents of phoma stem canker on oilseed rape, differ in their sensitivity to some azoles, which could be driving pathogen frequency change in crops. Here we used CYP51 protein modelling and heterologous expression to determine whether there are interspecific differences at the target-site level. Moreover, we provide an example of intrinsic sensitivity differences exhibited by both Leptosphaeria spp. in vitro and in planta. Comparison of homologous protein models identified highly conserved residues, particularly at the azole binding site, and heterologous expression of LmCYP51B and LbCYP51B, with fungicide sensitivity testing of the transformants, suggests that both proteins are similarly sensitive to azole fungicides flusilazole, prothioconazole-desthio and tebuconazole. Fungicide sensitivity testing on isolates shows that they sometimes have a minor difference in sensitivity in vitro and in planta. These results suggest that azole fungicides remain a useful component of integrated phoma stem canker control in the UK due to their effectiveness on both Leptosphaeria spp. Other factors, such as varietal resistance or climate, may be driving observed frequency changes between species

Proposal for a unified nomenclature for target site mutations associated with resistance to fungicides

Evolved resistance to fungicides is a major problem limiting our ability to control agricultural, medical and veterinary pathogens and is frequently associated with substitutions in the amino acid sequence of the target protein. The convention for describing amino-acid substitutions is to cite the wild type amino acid, the codon number and the new amino acid, using the one letter amino acid code. It has frequently been observed that orthologous amino acid mutations have been selected in different species by fungicides from the same mode of action class, but the amino acids have different numbers. These differences in numbering arise from the different lengths of the proteins in each species. The purpose of the current paper is to propose a system for unifying the labelling of amino acids in fungicide target proteins. To do this we have produced alignments between fungicide target proteins of relevant species fitted to a well-studied “archetype” species. Orthologous amino acids in all species are then assigned numerical “labels” based on the position of the amino acid in the archetype protein

Evaluation of a paper by Guarnaccia et al. (2017) on the first report of Phyllosticta citricarpa in Europe

27The Plant Health Panel reviewed the paper by Guarnaccia et al. (2017) and compared theirfindingswith previous predictions on the establishment ofPhyllosticta citricarpa. Four species ofPhyllostictawere found by Guarnaccia et al. (2017) in Europe.P. citricarpaandP. capitalensisare well-definedspecies, withP. citricarparecorded for thefirst time in Europe, confirming predictions by Magareyet al. (2015) and EFSA (2008, 2014, 2016) thatP. citricarpacan establish in some European citrus-growing regions. Two new speciesP. paracitricarpaandP. paracapitalensiswere also described, withP. paracitricarpa(found only in Greece) shown to be pathogenic on sweet orange fruits.Genotyping oftheP. citricarpaisolates suggests at least two independent introductions, with the population inPortugal being different from that present in Malta and Italy.P. citricarpaandP. paracitricarpawereisolated only from leaf litter in backyards. However, sinceP. citricarpadoes not infect or colonise deadleaves, the pathogen must have infected the above living leaves in citrus trees nearby. Guarnacciaet al. (2017) considered introduction to be a consequence ofP. citricarpahaving long been present orof illegal movement of planting material. In the Panel’s view, the fruit pathway would be an equally ormore likely origin. The authors did not report how surveys for citrus black spot (CBS) disease werecarried out, therefore their claim that there was no CBS disease even where the pathogen was presentis not supported by the results presented. From previous simulations, the locations where Guarnacciaet al. (2017) foundP. citricarpaorP. paracitricarpawere conducive forP. citricarpaestablishment, withnumber of simulated infection events by pycnidiospores comparable to sites of CBS occurrence outsideEurope. Preliminary surveys by National Plant Protection Organisations (NPPOs) have not confirmed sofar thefindings by Guarnaccia et al. (2017) but monitoring is still ongoingopenopenJeger, Michael; Bragard, Claude; Caffier, David; Candresse, Thierry; Chatzivassiliou, Elisavet; Dehnen‐Schmutz, Katharina; Gilioli, Gianni; Grégoire, Jean‐Claude; Jaques Miret, Josep Anton; MacLeod, Alan; Navajas Navarro, Maria; Niere, Björn; Parnell, Stephen; Potting, Roel; Rafoss, Trond; Rossi, Vittorio; Urek, Gregor; Van Bruggen, Ariena; Van Der Werf, Wopke; West, Jonathan; Winter, Stephan; Baker, Richard; Fraaije, Bart; Vicent, Antonio; Behring, Carsten; Mosbach Schulz, Olaf; Stancanelli, GiuseppeJeger, Michael; Bragard, Claude; Caffier, David; Candresse, Thierry; Chatzivassiliou, Elisavet; Dehnen‐schmutz, Katharina; Gilioli, Gianni; Grégoire, Jean‐claude; Jaques Miret, Josep Anton; Macleod, Alan; Navajas Navarro, Maria; Niere, Björn; Parnell, Stephen; Potting, Roel; Rafoss, Trond; Rossi, Vittorio; Urek, Gregor; Van Bruggen, Ariena; Van Der Werf, Wopke; West, Jonathan; Winter, Stephan; Baker, Richard; Fraaije, Bart; Vicent, Antonio; Behring, Carsten; Mosbach Schulz, Olaf; Stancanelli, Giusepp

Endophytic bacterial community composition in wheat (Triticum aestivum) is determined by plant tissue type, developmental stage and soil nutrient availability

Aims: To understand effects of tissue type, growth stage and soil fertilisers on bacterial endophyte communities of winter wheat (Triticum aestivum cv. Hereward). Methods: Endophytes were isolated from wheat grown under six fertiliser conditions in the long term Broadbalk Experiment at Rothamsted Research, UK. Samples were taken in May and July from root and leaf tissues. Results: Root and leaf communities differed in abundance and composition of endophytes. Endophytes were most abundant in roots and the Proteobacteria were most prevalent. In contrast, Firmicutes and Actinobacteria, the Gram positive phyla, were most prevalent in the leaves. Both fertiliser treatment and sample time influenced abundance and relative proportions of each phylum and genus in the endosphere. A higher density of endophytes was found in the Nil input treatment plants. Conclusions: Robust isolation techniques and stringent controls are critical for accurate recovery of endophytes. The plant tissue type, plant growth stage, and soil fertiliser treatment all contribute to the composition of the endophytic bacterial community in wheat. These results should help facilitate targeted development of endophytes for beneficial applications in agriculture

Sterol content analysis suggests altered eburicol 14 alpha-demethylase (CYP51) activity in isolates of Mycosphaerella graminicola adapted to azole fungicides

The recent decline in the effectiveness of some azole fungicides in controlling the wheat pathogen Mycosphaerella graminicola has been associated with mutations in the CYP51 gene encoding the azole target, the eburicol 14 alpha-demethylase (CYP51), an essential enzyme of the ergosterol biosynthesis pathway. In this study, analysis of the sterol content of M. graminicola isolates carrying different variants of the CYP51 gene has revealed quantitative differences in sterol intermediates, particularly the CYP51 substrate eburicol. Together with CYP51 gene expression studies, these data suggest that mutations in the CYP51 gene impact on the activity of the CYP51 protein

Evidence of Resistance to QoI Fungicides in Contemporary Populations of <i>Mycosphaerella fijiensis</i>, <i>M. musicola</i> and <i>M. thailandica</i> from Banana Plantations in Southeastern Brazil

Yellow and black Sigatoka, caused by Mycosphaerella fijiensis and M. musicola, respectively, are the most important worldwide foliar diseases of bananas. Disease control is heavily dependent on intensive fungicide sprays, which increase selection pressure for fungicide resistance in pathogen populations. The primary objective of this study was to assess the level and spread of resistance to quinone-outside inhibitors (QoI—strobilurin) fungicides in populations of both pathogens sampled from banana fields under different fungicide spray regimes in Southeastern Brazil. Secondly, we aimed to investigate when QoI resistance was confirmed if this was associated with the target-site alteration G143A caused by a mutation in the mitochondrial encoded cytochrome b gene. QoI resistance was detected in fungicide treated banana fields, while no resistance was detected in the organic banana field. A total of 18.5% of the isolates sampled from the pathogens’ populations were resistant to QoI. The newly described M. thailandica was also found. It was the second most abundant Mycosphaerella species associated with Sigatoka-like leaf spot symptoms in the Ribeira Valley and the highest level of QoI resistance was found for this pathogen. The G143A cytochrome b alteration was associated with the resistance to the QoI fungicides azoxystrobin and trifloxystrobin in M. fijiensis, M. musicola and M. thailandica strains. In order to reduce resistance development and maintain the efficacy of QoI fungicides, anti-resistance management strategies based on integrated disease management practices should be implemented to control the Sigatoka disease complex

Molecular Modelling of the Emergence of Azole Resistance in Mycosphaerella graminicola

A structural rationale for recent emergence of azole (imidazole and triazole) resistance associated with CYP51 mutations in the wheat pathogen Mycosphaerella graminicola is presented, attained by homology modelling of the wild type protein and 13 variant proteins. The novel molecular models of M. graminicola CYP51 are based on multiple homologues, individually identified for each variant, rather than using a single structural scaffold, providing a robust structure-function rationale for the binding of azoles, including important fungal specific regions for which no structural information is available. The wild type binding pocket reveals specific residues in close proximity to the bound azole molecules that are subject to alteration in the variants. This implicates azole ligands as important agents exerting selection on specific regions bordering the pocket, that become the focus of genetic mutation events, leading to reduced sensitivity to that group of related compounds. Collectively, the models account for several observed functional effects of specific alterations, including loss of triadimenol sensitivity in the Y137F variant, lower sensitivity to tebuconazole of I381V variants and increased resistance to prochloraz of V136A variants. Deletion of Y459 and G460, which brings about removal of that entire section of beta turn from the vicinity of the binding pocket, confers resistance to tebuconazole and epoxiconazole, but sensitivity to prochloraz in variants carrying a combination of A379G I381V ΔY459/G460. Measurements of binding pocket volume proved useful in assessment of scope for general resistance to azoles by virtue of their accommodation without bonding interaction, particularly when combined with analysis of change in positions of key amino acids. It is possible to predict the likely binding orientation of an azole molecule in any of the variant CYPs, providing potential for an in silico screening system and reliable predictive approach to assess the probability of particular variants exhibiting resistance to particular azole fungicides.</p

Mechanism of Binding of Prothioconazole to Mycosphaerella graminicola CYP51 Differs from That of Other Azole Antifungals ▿

Prothioconazole is one of the most important commercially available demethylase inhibitors (DMIs) used to treat Mycosphaerella graminicola infection of wheat, but specific information regarding its mode of action is not available in the scientific literature. Treatment of wild-type M. graminicola (strain IPO323) with 5 μg of epoxiconazole, tebuconazole, triadimenol, or prothioconazole ml−1 resulted in inhibition of M. graminicola CYP51 (MgCYP51), as evidenced by the accumulation of 14α-methylated sterol substrates (lanosterol and eburicol) and the depletion of ergosterol in azole-treated cells. Successful expression of MgCYP51 in Escherichia coli enabled us to conduct spectrophotometric assays using purified 62-kDa MgCYP51 protein. Antifungal-binding studies revealed that epoxiconazole, tebuconazole, and triadimenol all bound tightly to MgCYP51, producing strong type II difference spectra (peak at 423 to 429 nm and trough at 406 to 409 nm) indicative of the formation of classical low-spin sixth-ligand complexes. Interaction of prothioconazole with MgCYP51 exhibited a novel spectrum with a peak and trough observed at 410 nm and 428 nm, respectively, indicating a different mechanism of inhibition. Prothioconazole bound to MgCYP51 with 840-fold less affinity than epoxiconazole and, unlike epoxiconazole, tebuconazole, and triadimenol, which are noncompetitive inhibitors, prothioconazole was found to be a competitive inhibitor of substrate binding. This represents the first study to validate the effect of prothioconazole on the sterol composition of M. graminicola and the first on the successful heterologous expression of active MgCYP51 protein. The binding affinity studies documented here provide novel insights into the interaction of MgCYP51 with DMIs, especially for the new triazolinethione derivative prothioconazole