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Development of real-time PCR systems based on SYBR® Green I and TaqMan® technologies for specific quantitative detection of Phoma tracheiphila in infected Citrus

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Abstract

Real-time PCR assays based on SYBR® Green I and TaqMan® technologies were developed for in planta detection and quantification of Phoma tracheiphila, the mitosporic fungus causing ‘mal secco’ disease on citrus. Primers and a hybridization probe were designed on the basis of the internal transcribed spacer (ITS) region of the nuclear rRNA genes. The real-time PCR assays were compared with a classic isolation method in two separate experiments carried out on 6 and 24 month-old sour orange seedlings, artificially inoculated with a conidial suspension of the pathogen. Both technologies made it possible to follow the progression of infection by P. tracheiphila, enabling detection and quantification of the target fungus prior to the development of symptoms. The detection limit was 10 copies of the cloned target sequence and 15 pg of genomic DNA extracted from fungal spores. The values of the cycle threshold (Ct) were linearly correlated with the concentration of the target DNA, indicating that the method is suitable as a qualitative and quantitative assay. The presence of non-target fungal DNA had no effect on the specificity of the assay, but resulted in a 10-fold reduction of sensitivity. Total inhibition of the reaction occurred when conidia of the target pathogen were mixed with an organic soil substrate before extracting DNA using the standard protocol, while an alternative purification kit resulted in a significant decrease in sensitivity. Compared to classic methods, real-time PCR proved faster and easier to perform and showed a higher sensitivity. These results suggest that real-time PCR, based on both chemistries, has a great potential for early diagnosis of ‘mal secco’ disease and for quantitative estimation of fungal growth within host tissue.

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Acknowledgements

This work was funded by the European Union within the framework of INTERREG III A Italy–France–‘Isole’ (Project acronym: CITRUS). The authors wish to thank Franco Masia, Umberto Pirisi and Giuseppina Emonti for excellent technical assistance.

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Authors and Affiliations

  1. Unità di ricerca Istituto Nazionale Biostrutture e Biosistemi, Università di Sassari, Via E. De Nicola 9, 07100, Sassari, Italy

    Maria Antonietta Demontis, Marcella Orrù, Virgilio Balmas, Valentina Chessa, Laura Mascia & Quirico Migheli

  2. Dipartimento di Protezione delle Piante, Università di Sassari, Via E. De Nicola 9, 07100, Sassari, Italy

    Maria Antonietta Demontis, Marcella Orrù, Virgilio Balmas, Valentina Chessa, Laura Mascia & Quirico Migheli

  3. Dipartimento S.En.Fi.Mi.Zo., Università di Palermo, Viale delle Scienze 2, 90128, Palermo, Italy

    Santa Olga Cacciola

  4. Consiglio Nazionale delle Ricerche, Istituto di BioFisica, sede di Pisa, Area della Ricerca, Via Moruzzi 1, 56124, Pisa, Italy

    Bianca Elena Maserti & Laura Mascia

  5. Dipartimento di Gestione dei Sistemi Agrari e Forestali, Università Mediterranea di Reggio Calabria, Località Feo di Vito, 89060, Reggio Calabria, Italy

    Francesco Raudino & Gaetano Magnano di San Lio

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  1. Maria Antonietta Demontis
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  2. Santa Olga Cacciola
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  3. Marcella Orrù
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  4. Virgilio Balmas
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  6. Bianca Elena Maserti
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  7. Laura Mascia
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  9. Gaetano Magnano di San Lio
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  10. Quirico Migheli
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Correspondence to Quirico Migheli.

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Demontis, M.A., Cacciola, S.O., Orrù, M. et al. Development of real-time PCR systems based on SYBR® Green I and TaqMan® technologies for specific quantitative detection of Phoma tracheiphila in infected Citrus . Eur J Plant Pathol 120, 339–351 (2008). https://doi.org/10.1007/s10658-007-9222-9

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  • DOI: https://doi.org/10.1007/s10658-007-9222-9

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