Pfcyp51 exclusively determines reduced sensitivity to 14α-demethylase inhibitor fungicides in the banana black Sigatoka pathogen Pseudocercospora fijiensis.

Pablo Chong,Rafael E Arango Isaza,Aikaterini-Eleni Vichou,Gert H J Kema,Harold J G Meijer,Henk J Schouten,Beom Seok Kim

doi:10.1371/journal.pone.0223858

Abstract

The haploid fungus Pseudocercospora fijiensis causes black Sigatoka in banana and is chiefly controlled by extensive fungicide applications, threatening occupational health and the environment. The 14α-Demethylase Inhibitors (DMIs) are important disease control fungicides, but they lose sensitivity in a rather gradual fashion, suggesting an underlying polygenic genetic mechanism. In spite of this, evidence found thus far suggests that P. fijiensis cyp51 gene mutations are the main responsible factor for sensitivity loss in the field. To better understand the mechanisms involved in DMI resistance, in this study we constructed a genetic map using DArTseq markers on two F1 populations generated by crossing two different DMI resistant strains with a sensitive strain. Analysis of the inheritance of DMI resistance in the F1 populations revealed two major and discrete DMI-sensitivity groups. This is an indicative of a single major responsible gene. Using the DMI-sensitivity scorings of both F1 populations and the generation of genetic linkage maps, the sensitivity causal factor was located in a single genetic region. Full agreement was found for genetic markers in either population, underlining the robustness of the approach. The two maps indicated a similar genetic region where the Pfcyp51 gene is found. Sequence analyses of the Pfcyp51 gene of the F1 populations also revealed a matching bimodal distribution with the DMI resistant. Amino acid substitutions in P. fijiensis CYP51 enzyme of the resistant progeny were previously correlated with the loss of DMI sensitivity. In addition, the resistant progeny inherited a Pfcyp51 gene promoter insertion, composed of a repeat element with a palindromic core, also previously correlated with increased gene expression. This genetic approach confirms that Pfcyp51 is the single explanatory gene for reduced sensitivity to DMI fungicides in the analysed P. fijiensis strains. Our study is the first genetic analysis to map the underlying genetic factors for reduced DMI efficacy.

Highlights

The dothideomycete fungus Pseudocercospora fijiensis is the causal agent of black Sigatoka, a major global threat to banana crops that is responsible for serious economic losses in banana production and provokes major negative environmental impacts due to current control strategies [1]
Observed genetic mechanisms resulting in reduced Demethylase Inhibitors (DMIs) sensitivity in P. fijiensis are point mutations in and overexpression of 14α-demethylase that is encoded by the Pfcyp51 gene [5,6,7,8,9,10]
Successful crosses were accomplished after two experimental failures where we empirically determined the critical number of sub-cultivations of the parental strains, which should not be more than two in order to maintain sexual fitness

Summary

Introduction

The dothideomycete fungus Pseudocercospora fijiensis (previously Mycosphaerella fijiensis) is the causal agent of black Sigatoka, a major global threat to banana crops that is responsible for serious economic losses in banana production and provokes major negative environmental impacts due to current control strategies [1]. Contemporary disease control is mainly achieved by the application of systemic fungicides of which the most commonly used fungicides belong to the 14α-Demethylase Inhibitors (DMIs) group. DMI are single target fungicides, sensitive to resistance development. Fungicide application frequencies for black Sigatoka management are extensive and DMIs are important constituents of the spray schedules, which have serious negative environmental and social impacts, and contribute to the development of resistance in the pathogen populations [2,3,4]. Observed genetic mechanisms resulting in reduced DMI sensitivity in P. fijiensis are point mutations in and overexpression of 14α-demethylase that is encoded by the Pfcyp gene [5,6,7,8,9,10]

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Conclusion