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Quantitative PCR for detection and quantification of Phytophthora cactorum in the cultivation of strawberry

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Abstract

In this study, we report on the development of two quantitative PCR assays for the detection and quantification of the soilborne plant pathogen Phytophthora cactorum causing root and crown rot of strawberry. The assays rely on the use of SYBR Green I chemistry and use the internal transcribed spacer region 2 (ITS2) and the Ras-related protein gene Ypt1 as detection markers. An extensive list of Phytophthora isolates was included in the study to evaluate assay specificity. High specificity was obtained when Ypt1 was used as detection marker, with cross-reactions only occurring with the closest relatives of P. cactorum, including P. hedraiandra, P. idaei and P. pseudotsugae. In contrast, the ITS-based assay was less specific, resulting in a larger number of cross-reactions (eight species when tested at 10 pg DNA). In sensitivity tests, P. cactorum DNA was detected down to 10 fg using the ITS-based assay, while the limit of detection (LOD) for the Ypt1-based assay was 1 pg DNA, irrespective of the presence of background DNA from strawberry or soil. When strawberry, growth substrate and soil samples were spiked with a 10-fold dilution series of P. cactorum zoospores, the LOD for the ITS-based assay was 10 zoospores g−1 plant material, growth substrate or soil. The limit of quantification (LOQ) of the assay was 109 zoospores g−1 plant material, 15.8 zoospores g−1 growth substrate, and 106 zoospores g−1 soil. For the Ypt1-based assay, the LOD and LOQ values were 1000 and 5910 zoospores g−1 plant material, 1000 and 1818 zoospores g−1 growth substrate, and 1000 and 3823 zoospores g−1 soil, respectively. In terms of detection, analysis of field samples suggested that the Ypt1-based assay almost performed equally well compared to the ITS-based assay, especially for plant and growth substrate samples. Further, our results showed that both qPCR assays outperformed classical plating, illustrating their power to be used for diagnostic purposes. Interestingly, P. cactorum was also detected in soil and growth substrate samples from areas with no symptoms, suggesting that the assays can also aid in early pathogen detection before the disease manifests.

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Acknowledgments

We would like to thank VLAIO (Flanders Innovation and Entrepreneurship) for financial support (HBC.2017.0833). Additionally, we are grateful to our colleagues W. Man in ‘t Veld, A. Numansen, L. Kroon, W. Flier, F. Govers, D. Cooke, K. Hughes, S. Werres, J. Bakyoni, S. Klemsdal, E. Hansen and M. Coffey for giving us access their Phytophthora collections. Further, we would like to thank all growers providing samples for this research.

Funding

This study was funded by VLAIO (Flanders Innovation and Entrepreneurship), project HBC.2017.0833.

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Authors and Affiliations

  1. CMPG Laboratory for Process Microbial Ecology and Bioinspirational Management (PME&BIM), Department of Microbial and Molecular Systems, KU Leuven, Willem De Croylaan 46, box 2458, B-3001, Leuven, Belgium

    E. Verdecchia, J. Ferreira, H. Rediers & B. Lievens

  2. Leuven Plant Institute (LPI), B-3001, Leuven, Belgium

    E. Verdecchia, J. Ferreira, H. Rediers & B. Lievens

  3. Research Center for Fruit Growing, B-3800, Sint-Truiden, Belgium

    A. Ceustermans, W. Van Hemelrijck & D. Bylemans

  4. Research Center Hoogstraten, B-2328, Meerle, Belgium

    D. Baets & P. Melis

  5. Wageningen University and Research, PB 6708, Wageningen, The Netherlands

    P. Bonants

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  1. E. Verdecchia
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  2. A. Ceustermans
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  3. D. Baets
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  4. J. Ferreira
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  6. P. Melis
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  9. H. Rediers
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  10. B. Lievens
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Contributions

EV, HR and BL conceived the ideas and designed methodology. EV, AC, DB and JF collected the data. PM, WVH, HR and BL contributed to infrastructure, equipment, and reagents. PB provided DNA samples. EV, HR and BL analyzed the data. EV and BL led the writing of the manuscript. All authors contributed critically to the drafts of this manuscript and gave final approval for publication.

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Correspondence to B. Lievens.

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Rediers, H. and Lievens, B. are shared last co-author

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Verdecchia, E., Ceustermans, A., Baets, D. et al. Quantitative PCR for detection and quantification of Phytophthora cactorum in the cultivation of strawberry. Eur J Plant Pathol 160, 867–882 (2021). https://doi.org/10.1007/s10658-021-02290-z

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