Abstract
Downy mildew is a major disease of boysenberries in New Zealand, caused by Peronospora sparsa. Most boysenberry plant material, including tissue culture propagated plants are systemically infected and this pathogen also presents as a latent infection. The current nested PCR method to detect latent infection of P. sparsa in asymptomatic boysenberry plants is time consuming as it employs two separate PCR, with potential contamination producing false positive and/or false negative results. To overcome these issues a one step nested PCR method was developed. The method was optimised for primer concentrations, PCR cycle number and DNA concentration. DNA was extracted using a CTAB method. The one step nested PCR method could detect latent infection of P. sparsa in both dormant and active plant growth at 0.4 pg genomic DNA. The most reliable detection was achieved from crown or root tissues. For surety of the infection status, replicate plant tissues should be assessed by PCR as inconsistency between the one step nested PCR and fluorescence microscopy indicated that P. sparsa colonisation is discontinuous through the plant. This method can be recommended for screening P. sparsa latent infection in boysenberry mother plants and daughter plants in nurseries, due to high sensitivity, improved throughput, low cost, and low contamination risk.
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Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.
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Acknowledgements
This research was funded by the New Zealand Ministry of Science and Innovation, and the Boysenberry Council Ltd. Mr. Julian Raine and New Zealand boysenberry growers gave valuable support. Mr. Geoff Langford provided advice on growing plants. In addition Mr. Brent Richards and Mrs. Leona Meachen provided assistance in maintenance of plants in the nursery.
Funding
Funding was provided by New Zealand Ministry of Science and Innovation (Postgraduate research scholarship awarded to the first author) and Boysenberry Council Ltd.
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All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Anusara Herath Mudiyanselage and Hayley Ridgway. The first draft of the manuscript was written by Anusara Herath Mudiyanselage and Eirian Jones. All authors revised all previous versions of the manuscript.
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Mudiyanselage, A.M.H., Jones, E.E., Jaspers, M.V. et al. A one step nested PCR method for detection of Peronospora sparsa, the downy mildew pathogen, in boysenberry (Rubus ursinus). Eur J Plant Pathol 160, 973–981 (2021). https://doi.org/10.1007/s10658-021-02283-y
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DOI: https://doi.org/10.1007/s10658-021-02283-y