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New multiplex conventional PCR and quadruplex real-time PCR assays for one-tube detection of Phyllosticta citricarpa, Elsinoë fawcettii, Elsinoë australis, and Pseudocercospora angolensis in Citrus: development and validation

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Abstract

Phyllosticta citricarpa, Elsinoë fawcettii, Elsinoë australis, and Pseudocercospora angolensis are major pathogens of citrus crops worldwide and can cause non-characteristic symptoms that may lead to confusion regarding the causative agent. These fungi are subject to international phytosanitary regulations, and testing on fruits or leaves requires accurate and easy-to-use tools. New multiplex conventional PCR and real-time PCR assays were developed here to achieve highly accurate simultaneous detection of all four fungal pathogens in fruit tissues. We designed new oligonucleotide combinations for P. citricarpa, E. fawcettii, and E. australis and combined them with already available primers and hydrolysis probes to be used in either PCR assay. The limit of detection for multiplex conventional PCR was as low as 100 pg μL−1 for P. citricarpa, E. fawcettii, and E. australis and 10 pg μL−1 of target DNA per reaction tube for P. angolensis. The quadruplex real-time PCR assay successfully yielded repeatable positive results with as low as 242, 243, 241, and 242 plasmidic copies of target DNA of P. citricarpa, E. fawcettii, E. australis, and P. angolensis, respectively. Moreover, analysis of 60 naturally infected citrus samples yielded 100% concordant results by both assays. Our validation experiment revealed that the multiplex real-time PCR assay showed high specificity except a cross-reaction with P. paracitricarpa DNA. Sensitivity, repeatability, reproducibility, and robustness were verified, and the assay could be used following different DNA extraction procedures, supporting fitness for routine analysis. These new multiplex tools should be of great interest as cost-effective solutions for regulatory authorities and diagnostic laboratories, enabling testing for four important pathogens in single-tube reactions.

Key points

• Development of new conventional PCR and qPCR assays for four citrus pathogens.

• Very low limits of detection were found for multiplex conventional PCR.

• qPCR had high specificity, sensitivity, repeatability, reproducibility, and robustness.

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Acknowledgments

This research was part of the PACE2D research project in the framework of the PHC IMHOTEP (France/Egypt) program and was funded by an Academic and scientific research and technology grant from the French Embassy in Egypt (IFE), (Campus France project ID 37589QE), the French Agency for Food, Environmental and Occupational Health and Safety (ANSES), and the Agricultural Research Centre (ARC, Egypt). The authors wish to thank Isabelle Cerf-Wendling for her assistance, Cécile Guinet (ANSES), for her technical and scientific support, and Pascal Frey for his assistance in DNA extraction experiments, as well as Blanca Isabel Canteros (EEA INTA) for sending citrus samples from Argentina.

Author contribution statement

YA, AH, JA, and RI conceived and designed the research. Material preparation and data collection were performed by YA, AH, JH, and CFJ. YA and AH analyzed the data. YA, AH, and RI wrote the manuscript, and all authors commented on previous versions of the manuscript. All authors read and approved the manuscript.

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Authors and Affiliations

  1. Plant Quarantine Pathogens Laboratory (PQPL), Plant Pathology Research Institute, Agricultural Research Center, 9 Gamaa Street, Giza, Egypt

    Yosra Ahmed

  2. ANSES Laboratoire de la Santé des Végétaux, Unité de Mycologie, Domaine de Pixérécourt, 54220, Malzéville, France

    Yosra Ahmed, Jacqueline Hubert, Céline Fourrier-Jeandel, Jaime Aguayo & Renaud Ioos

  3. Central Administration of Plant Quarantine, Ministry of Agriculture and Land Reclamation, Nadi ElSaid Street, Dokki, Giza, 12618, Egypt

    Ahmed Hussein

  4. Regional Office for Near East and North Africa, Food and Agriculture Organization of the United Nations, Dokki, Giza, Egypt

    Ahmed Hussein

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  1. Yosra Ahmed
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  2. Ahmed Hussein
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  3. Jacqueline Hubert
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  4. Céline Fourrier-Jeandel
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  5. Jaime Aguayo
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  6. Renaud Ioos
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Correspondence to Renaud Ioos.

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Ahmed, Y., Hussein, A., Hubert, J. et al. New multiplex conventional PCR and quadruplex real-time PCR assays for one-tube detection of Phyllosticta citricarpa, Elsinoë fawcettii, Elsinoë australis, and Pseudocercospora angolensis in Citrus: development and validation. Appl Microbiol Biotechnol 104, 9363–9385 (2020). https://doi.org/10.1007/s00253-020-10880-w

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