Development of a Highly Sensitive FcMito qPCR Assay for the Quantification of the Toxigenic Fungal Plant Pathogen Fusarium culmorum.

Katarzyna Bilska,Dariusz Załuski,Maciej Buśko,Anna Baturo-Cieśniewska,Marco Beyer,Tomasz Kulik,Matias Pasquali,Kinga Treder,Anna Ostrowska-Kołodziejczak,Juliusz Perkowski,Joerg Denekas,Marcin Juda

doi:10.3390/toxins10050211

Abstract

Fusarium culmorum is a ubiquitous, soil-borne fungus (ascomycete) causing foot and root rot and Fusarium head blight on cereals. It is responsible for yield and quality losses as well as grain contamination with mycotoxins, which are a potential health hazard. An extremely sensitive mitochondrial-based qPCR assay (FcMito qPCR) for quantification of F. culmorum was developed in this study. To provide specificity, the FcMito assay was successfully validated against 85 F. culmorum strains and 53 isolates of 30 other fungal species. The assay efficiency and sensitivity were evaluated against different F. culmorum strains with various amounts of pure fungal DNA and in the presence of background wheat DNA. The results demonstrated the high efficiency of the assay (97.2–106.0%, R2-values > 0.99). It was also shown that, in the presence of background DNA, 0.01 pg of fungal template could be reliably quantified. The FcMito assay was used to quantify F. culmorum DNA using 108 grain samples with different trichothecene levels. A significant positive correlation was found between fungal DNA quantity and the total trichothecene content. The obtained results showed that the sensitivity of the FcMito assay was much higher than the nuclear-based qPCR assay for F. culmorum.

Highlights

A common soil-borne fungus, Fusarium culmorum, remains an important cereal pathogen of a wide range of small-grain cereals, as well as maize [1,2]
The Design of a Primer/Probe Set for Specific Quantification of F. culmorum Based on the Mitochondrial
An intron3 within the COX2 gene was present in F. culmorum only, based on which a primer/probe set was designed for specific quantification of this species

Summary

Introduction

A common soil-borne fungus, Fusarium culmorum, remains an important cereal pathogen of a wide range of small-grain cereals, as well as maize [1,2]. It causes two distinct diseases: FRR (Fusarium foot and root rot) and FHB (Fusarium head blight), known as ear blight or scab [1]. Both diseases cause significant yield losses and the latter results in contamination of the grain with trichothecenes, which may pose a grave threat to both food and feed safety [3,4,5,6]. F. culmorum can be controlled by fungicides containing triazoles as active ingredients, while strobilurins such as trifloxystrobin and succinate dehydrogenase inhibitors such as isopyrazam are hardly effective [9]

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Results

Conclusion