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Multiplex real-time PCR for detection and quantification of Colletotrichum abscissum and C. gloeosporioides on Citrus leaves

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Abstract

Colletotrichum abscissum and C. gloeosporioides are the causal agents of citrus Postbloom Fruit Drop (PFD), a major disease in several countries of the American continent. These pathogens infect only floral structures; however, in the absence of floral tissue, they survive asymptomatically on the leaf tissue. Thus, detecting and quantifying pathogens on leaves are critical for epidemiological studies. Colletotrichum species are detected through isolation, serological methods and conventional PCR, but these techniques are not effective for accurate detection of PFD agents. This study aimed to develop specific, more sensitive, faster and less laborious techniques than the traditional ones routinely used. We developed, standardized and validated a multiplex real-time PCR with high sensitivity (98–99.3%) and specificity (94.3–97.1%) for the detection and quantification of both pathogens. This technique was 1000 and 10,000 times more sensitive than Nested-PCR and PCR, respectively for the detection of C. abscissum. Similarly, the multiplex qPCR was 100 and 1000 times more sensitive than Nested-PCR and PCR, respectively for the detection of C. gloeosporioides. The diagnostic parameters used to validate the multiplex qPCR showed a high positive (17–35) and low negative (0.006–0.95) likelihood ratios, low percentage of false positives (2.8–5.6%) and false negatives (0.6–1.9%), good agreement between the results generated between qPCR and multiplex qPCR, revealed by the Kappa index (0.91–0.98) and coincidental results (95–99%). All parameters indicated that the multiplex qPCR was effectively validated to detect and quantify the causal agents of PFD.

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Acknowledgments

This research was supported by a grant from São Paulo Research Foundation (FAPESP 2011/11629-1), National Council for Scientific and Technological Development (CNPq-140227/2013-0) and Coordination for the Improvement of Higher Education Personnel (CAPES-9810-11-0). We thank Dr. Antonio Vicent Civera for allowing the use of the mycology laboratory Valencian Institute for Agricultural Research to conduct some experiments.

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Authors and Affiliations

  1. Center for Plant Protection and Biotechnology, Valencian Institute for Agricultural Research (IVIA), Carretera Moncada-Náquera, Km 5, 46113, Moncada, Valencia, Spain

    W. V. Pereira, E. Bertolini & M. Cambra

  2. Department of Plant Sciences and Crop Protection, Federal University of Paraná, R. Funcionários 1540, Curitiba, PR, 80.035-050, Brazil

    W. V. Pereira

  3. Department of Plant Protection, Faculty of Agronomy, Federal University of Rio Grande do Sul, Avenida Bento Gonçalves 7712, Porto Alegre, RS, 91540-000, Brazil

    E. Bertolini

  4. Department of Plant Pathology and Nematology, University of São Paulo/ESALQ, Avenida Pádua Dias 11, Piracicaba, SP, 13418-900, Brazil

    N. S. Massola Junior

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  1. W. V. Pereira
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  2. E. Bertolini
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  3. M. Cambra
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  4. N. S. Massola Junior
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Correspondence to W. V. Pereira.

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Pereira, W.V., Bertolini, E., Cambra, M. et al. Multiplex real-time PCR for detection and quantification of Colletotrichum abscissum and C. gloeosporioides on Citrus leaves. Eur J Plant Pathol 155, 1047–1059 (2019). https://doi.org/10.1007/s10658-019-01831-x

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  • DOI: https://doi.org/10.1007/s10658-019-01831-x

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