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A portable recombinase polymerase amplification assay for the rapid detection of cucurbit leaf crumple virus in watermelon leaves and fruits

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Abstract

Cucurbit leaf crumple virus (CuLCrV), a single-stranded DNA virus belonging to the species Cucurbit leaf crumple virus in the genus Begomovirus, causes cucurbit leaf crumple disease in many species of the Cucurbitaceae family. This report describes the development and standardization of field-based isothermal recombinase polymerase amplification (RPA) and exonuclease RPA (exo-RPA) assays for the detection and diagnosis of CuLCrV in the leaves of watermelon, squash and pumpkin, and the fruits of watermelon. The oligonucleotide primers were made to target a portion of the BL1 movement protein. The primer set used in the RPA was highly specific for CuLCrV and did not show a cross-reaction with other cucurbit viruses nor in tissues with co-infections of CuLCrV with cucurbit yellow stunting disorder virus (CYSDV) and/or squash vein yellowing virus (SqVYV). These assays were sensitive, producing a positive signal with 1 fg/uL of purified total DNA and at a dilution of 1 × 10–5 of crude extract from CuLCrV-infected watermelon plants. Incorporation of a 6-fluorescein amidite (6-FAM) probe into the exo-RPA assay enabled a total sample processing and detection time of 30 min and positive reactions were detectable in different tissues including watermelon leaves, fruits, and pips (seeds lacking an embryo). The exo-RPA was efficiently used in the detection of CuLCrV in 21 cucurbit plants growing in different regions of southeastern U.S. A sensitive, rapid and easy field-based method for the detection and diagnosis of CuLCrV in watermelon was developed.

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Acknowledgements

This study was funded by the Florida Watermelon Association (FWA). The authors are thankful to Laura Land of the FWA for continued support, Dr. Babu Srinivasan and Dr. Bhabesh Dutta at the University of Georgia for sample collection in Georgia, and Dr. Scott Adkins, USDA-ARS for the valuable suggestions on the first draft of this manuscript.

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Authors and Affiliations

  1. Institute of Food and Agricultural Sciences (UF-IFAS), University of Florida, North Florida Research and Education Center, Quincy, FL, 32351, USA

    Melanie Kalischuk, Josh Freeman & Mathews Paret

  2. Department of Plant Agriculture, University of Guelph, Guelph, ON, N1G 2W1, Canada

    Melanie Kalischuk

  3. Southwest Florida Research and Education Center, UF-IFAS, Immokalee, FL, 34142, USA

    Katherine Hendricks & Pamela D. Roberts

  4. North Florida Research and Education Center, UF-IFAS, Live Oak, FL, 32060, USA

    Robert Hochmuth

  5. Department of Plant Pathology, University of Florida, Gainesville, FL, 32611, USA

    Josh Freeman, Pamela D. Roberts & Mathews Paret

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  1. Melanie Kalischuk
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Correspondence to Melanie Kalischuk or Mathews Paret.

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Kalischuk, M., Hendricks, K., Hochmuth, R. et al. A portable recombinase polymerase amplification assay for the rapid detection of cucurbit leaf crumple virus in watermelon leaves and fruits. J Plant Pathol 104, 215–224 (2022). https://doi.org/10.1007/s42161-021-00973-4

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  • DOI: https://doi.org/10.1007/s42161-021-00973-4

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