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Detection and quantification of Fusarium oxysporum f. sp. cucumerinum in environmental samples using a specific quantitative PCR assay

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Abstract

The rapid and reliable identification and quantification of pathogens is essential for the management of economically important plant diseases. Fusarium oxysporum f. sp. cucumerinum is the soil borne fungus responsible for Fusarium vascular wilt of cucumber. In this study, we report the development of a specific and reliable real-time quantitative PCR assay and the development of an ultra-sensitive diagnostic pseudo-nested PCR assay. The capacity of the PCR assays to accurately identify and quantify Fusarium oxysporum f. sp. cucumerinum was experimentally tested by the development of standard curves from serial dilutions of copy numbers in a range of complex environmental DNA samples. The amplification efficiency, sensitivity and reproducibility of the qPCR assays were not significantly affected by the presence of any of the non-target background DNA tested. In quantitative real-time PCR, as few as 100 copies could be reliably quantified, and in simple and pseudo-nested PCR as little as 10 pg and 10 fg, respectively, could be detected. This rapid and sensitive qPCR method can be used to facilitate investigations into plant–pathogen interactions, epidemiology, and disease management practices.

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Acknowledgments

The authors would like to acknowledge and thank the Royal Botanic Gardens and Department of Primary Industries NSW for access and use of their Fusarium Collections and Dr. Neil Wilson, Faculty of Agriculture and Environment, University of Sydney, for technical assistance.

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Authors and Affiliations

  1. Faculty of Agriculture and Environment, The University of Sydney, Biomedical Building C81, Sydney, New South Wales,, 2006, Australia

    Kelly Scarlett & David Guest

  2. Department of Primary Industries, Elizabeth Macarthur Agricultural Institute, Menangle, New South Wales, Australia

    Len Tesoriero & Rosalie Daniel

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  1. Kelly Scarlett
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  2. Len Tesoriero
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  3. Rosalie Daniel
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  4. David Guest
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Correspondence to Kelly Scarlett.

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Scarlett, K., Tesoriero, L., Daniel, R. et al. Detection and quantification of Fusarium oxysporum f. sp. cucumerinum in environmental samples using a specific quantitative PCR assay. Eur J Plant Pathol 137, 315–324 (2013). https://doi.org/10.1007/s10658-013-0244-1

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