Development of a Highly Sensitive FcMito qPCR Assay for the Quantification of the Toxigenic Fungal Plant Pathogen Fusarium culmorum
Katarzyna Bilska,
Tomasz Kulik,
Anna Ostrowska-Kołodziejczak,
Maciej Buśko,
Matias Pasquali,
Marco Beyer,
Anna Baturo-Cieśniewska,
Marcin Juda,
Dariusz Załuski,
Kinga Treder,
Joerg Denekas,
Juliusz Perkowski
Affiliations
Katarzyna Bilska
Department of Microbiology and Mycology, University of Warmia and Mazury in Olsztyn, Oczapowskiego 1A, 10-719 Olsztyn, Poland
Tomasz Kulik
Department of Microbiology and Mycology, University of Warmia and Mazury in Olsztyn, Oczapowskiego 1A, 10-719 Olsztyn, Poland
Anna Ostrowska-Kołodziejczak
Department of Chemistry, Poznań University of Life Sciences, Wojska Polskiego 75, 60-625 Poznań, Poland
Maciej Buśko
Department of Chemistry, Poznań University of Life Sciences, Wojska Polskiego 75, 60-625 Poznań, Poland
Matias Pasquali
Department of Food, Environmental and Nutritional Sciences (DEFENS), University of Milan, via Celoria 2, 20133 Milano, Italy
Marco Beyer
Department Environmental Research and Innovation, Luxembourg Institute of Science and Technology, 41, rue du Brill, L-4422 Belvaux, Luxembourg
Anna Baturo-Cieśniewska
Faculty of Agriculture and Biotechnology, Department of Phytopathology and Molecular Mycology, University of Technology and Life Sciences, Kordeckiego St. 20, 85-225 Bydgoszcz, Poland
Marcin Juda
Faculty of Agriculture and Biotechnology, Department of Phytopathology and Molecular Mycology, University of Technology and Life Sciences, Kordeckiego St. 20, 85-225 Bydgoszcz, Poland
Dariusz Załuski
Department of Plant Breeding and Seed Production, University of Warmia and Mazury in Olsztyn, Plac Łódzki 3, 10-727 Olsztyn, Poland
Kinga Treder
Department of Agroecosystems, University of Warmia and Mazury in Olsztyn, Plac Łódzki 3, 10-727 Olsztyn, Poland
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.
