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.
Similar content being viewed by others
References
Ahmed Y, Hubert J, Fourrier-Jeandel C, Dewdney MM, Aguayo J, Ioos R (2019) A set of conventional and multiplex real-time PCR assays for direct detection of Elsinoe fawcettii, E. australis, and Pseudocercospora angolensis in citrus fruits. Plant Dis 103(2):345–356. https://doi.org/10.1094/pdis-05-18-0798-re
Anonymous (2009) PM 7/17(2): Guignardia citricarpa. EPPO Bull 39:318–327
Anonymous (2019) Commission Implementing Regulation (EU) 2019/2072 of 28 November 2019 establishing uniform conditions for the implementation of Regulation (EU) 2016/2031 of the European Parliament and the Council, as regards protective measures against pests of plants, and repealing Commission Regulation (EC) No 690/2008 and amending Commission Implementing Regulation (EU) 2018/2019. OJ L 319:1–279
Bonants PJM, Carroll GC, Weerdt dM, Brouwershaven VIR, Baayen RP (2003) Development and validation of a fast PCR-based detection method for pathogenic isolates of the citrus black spot fungus, Guignardia citricarpa. Eur J Plant Pathol 109(5):503–513
Boughalleb-M’Hamdi N, Fathallah A, Benfradj N, Ben Mahmoud S, Bel Hadj Ali A, Medhioub L, Jaouadi I, Huber J, Jeandel C, Ioos R (2020) First report of citrus black spot disease caused by Phyllosticta citricarpa on Citrus limon and C. sinensis in Tunisia. New Dis Rep 41:8
Brentu FC, Cornelius EW, Lawson LEV, Oduro KA, Vicent A (2013) First report of Pseudocercospora angolensis causing fruit and leaf spot of citrus in Ghana. Plant Dis 97(12):1661–1661. https://doi.org/10.1094/PDIS-06-13-0615-PDN
Briggs RJ, Nicholson R, Vazvaei F, Busch J, Mabuchi M, Mahesh KS, Brudny-Kloeppel M, Weng N, Galvinas PAR, Duchene P, Hu P, Abbott RW (2014) Method transfer, partial validation, and cross validation: recommendations for best practices and harmonization from the global bioanalysis consortium harmonization team. AAPS J 16(6):1143–1148. https://doi.org/10.1208/s12248-014-9650-3
Broeders S, Huber I, Grohmann L, Berben G, Taverniers I, Mazzara M, Roosens N, Morisset D (2014) Guidelines for validation of qualitative real-time PCR methods. Trends Food Sci Tech 37(2):115–126. https://doi.org/10.1016/j.tifs.2014.03.008
Carbone I, Kohn LM (1999) A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia 91(3):553–556. https://doi.org/10.1080/00275514.1999.12061051
Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull 19:11–15
Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32(5):1792–1797. https://doi.org/10.1093/nar/gkh340
EFSA Panel on Plant Health (2014) Scientific opinion on the risk of Phyllosticta citricarpa (Guignardia citricarpa) for the EU territory with identification and evaluation of risk reduction options. EFSA J 12(2):3557. https://doi.org/10.2903/j.efsa.2014.3557
EFSA Panel on Plant Health (2017) Pest categorisation of Elsinoë fawcettii and E. australis. EFSA J 15(12):5100. https://doi.org/10.2903/j.efsa.2017.5100
EPPO (2010) PM 7/98 (1): specific requirements for laboratories preparing accreditation for a plant pest diagnostic activity. EPPO Bull 40(1):5–22. https://doi.org/10.1111/j.1365-2338.2009.02347.x
EPPO (2018) PM 7/76 (5) Use of EPPO diagnostic standards. EPPO Bull 48(3):373–377. https://doi.org/10.1111/epp.12506
EPPO (2020) EPPO global database. Phyllosticta citricarpa. https://gd.eppo.int/taxon/GUIGCI/distribution/TN. PUblisher
Glienke C, Pereira OL, Stringari D, Fabris J, Kava-Cordeiro V, Galli-Terasawa L, Cunnington J, Shivas RG, Groenewald JZ, Crous PW (2011) Endophytic and pathogenic Phyllosticta species, with reference to those associated with citrus black spot. Persoonia 26:47–56. https://doi.org/10.3767/003158511X569169
Guarnaccia V, Gehrmann T, Silva-Junior GJ, Fourie PH, Haridas S, Vu D, Spatafora J, Martin FM, Robert V, Grigoriev IV, Groenewald JZ, Crous PW (2019) Phyllosticta citricarpa and sister species of global importance to Citrus. Mol Plant Pathol 20(12):1619–1635. https://doi.org/10.1111/mpp.12861
Guarnaccia V, Groenewald JZ, Li H, Glienke C, Carstens E, Hattingh V, Fourie PH, Crous PW (2017) First report of Phyllosticta citricarpa and description of two new species, P. paracapitalensis and P. paracitricarpa, from citrus in Europe. Stud Mycol 87:161–185. https://doi.org/10.1016/j.simyco.2017.05.003
Hou X, Huang F, Zhang TY, Li HY (2013) Detection of Elsinoe fawcettii in planta through real-time PCR. J Food Agric Environ 11(3):1085–1087
Hyun JW, Yi SH, MacKenzie SJ, Timmer LW, Kim KS, Kang SK, Kwon HM, Lim HC (2009) Pathotypes and genetic relationship of worldwide collections of Elsinoë spp. causing scab diseases of Citrus. Phytopathology 99(6):721–728. https://doi.org/10.1094/phyto-99-6-0721
Ioos R, Aloi F, Piškur B, Cc G, Mullett M, Mn B, Bragança H, Cacciola SO, Oskay F, Cornejo C, Adamson K, Douanla-Meli C, Kačergius A, Martínez-Álvarez P, Nowakowska JA, Luchi N, Vettraino AM, Ahumada R, Pasquali M, Fourie G, Kanetis L, Alves A, Ghelardini L, DvořÁk M, Sanz-Ros A, Diez JJ, Baskarathevan J, Aguayo J (2019) Transferability of PCR-based diagnostic protocols: an international collaborative case study assessing protocols targeting the quarantine pine pathogen Fusarium circinatum. Sci Rep 9(1):1–17
Ioos R, Fourrier C, Iancu G, Gordon TR (2009) Sensitive detection of Fusarium circinatum in pine seed by combining an enrichment procedure with a real-time polymerase chain reaction using dual-labeled probe chemistry. Phytopathology 99(5):582–590. https://doi.org/10.1094/phyto-99-5-0582
Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, Thierer T, Ashton B, Meintjes P, Drummond A (2012) Geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28(12):1647–1649. https://doi.org/10.1093/bioinformatics/bts199
Kostov K, Verstappen ECP, Bergervoet JHW, de Weerdt M, Schoen CD, Slavov S, Bonants PJM (2016) Multiplex detection and identification of Phytophthora spp. using target-specific primer extension and Luminex xTAG technology. Plant Pathol 65(6):1008–1021. https://doi.org/10.1111/ppa.12481
Li R, Mock R, Huang Q, Abad J, Hartung J, Kinard G (2008) A reliable and inexpensive method of nucleic acid extraction for the PCR-based detection of diverse plant pathogens. J Virol Methods 154(1–2):48–55. https://doi.org/10.1016/j.jviromet.2008.09.008
Meyer L, Jacobs R, Korsten L, Kotze JM, Truter M (2012) Detection and molecular identification protocols for Phyllosticta citricarpa from citrus matter. S Afr J Sci 108(3–4):3–4
Meyer L, Sanders GM, Jacobs R, Korsten L (2006) A one-day sensitive method to detect and distinguish between the citrus black spot pathogen Guignardia citricarpa and the endophyte Guignardia mangiferae. Plant Dis 90(1):97–101. https://doi.org/10.1094/pd-90-0097
O’Donnell K, Kistler HC, Cigelnik E, Ploetz RC (1998) Multiple evolutionary origins of the fungus causing Panama disease of banana: concordant evidence from nuclear and mitochondrial gene genealogies. Proc Natl Acad Sci U S A 95(5):2044–2049. https://doi.org/10.1073/pnas.95.5.2044
Paul I, van Jaarsveld AS, Korsten L, Hattingh V (2005) The potential global geographical distribution of citrus black spot caused by Guignardia citricarpa (Kiely): likelihood of disease establishment in the European Union. Crop Prot 24(4):297–308
Peres NA, Harakava R, Carroll GC, Adaskaveg JE, Timmer LW (2007) Comparison of molecular procedures for detection and identification of Guignardia citricarpa and G. mangiferae. Plant Dis 91(5):525–531
Pretorius MC, Crous PW, Groenewald JZ, Braun U (2003) Phylogeny of some cercosporoid fungi from Citrus. Sydowia 55:286–305
Raja HS, Hustad CL, Shearer V, Carol Miller A (2011) Testing the phylogenetic utility of MCM7 in the Ascomycota. MycoKeys 1:63–94. https://doi.org/10.3897/mycokeys.1.1966
Schirmacher AM, Tomlinson JA, Barnes AV, Barton VC (2019) Species-specific real-time PCR for diagnosis of Phyllosticta citricarpa on Citrus species. EPPO Bull 49(2):306–313
Schmitt I, Crespo A, Divakar PK, Fankhauser JD, Herman-Sackett E, Kalb K, Nelsen MP, Nelson NA, Rivas-Plata E, Shimp AD, Widhelm T, Lumbsch HT (2009) New primers for promising single-copy genes in fungal phylogenetics and systematics. Persoonia 23:35–40. https://doi.org/10.3767/003158509X470602
Tan MK, Timmer LW, Broadbent P, Priest M, Cain P (1996) Differentiation by molecular analysis of Elsinoe spp. causing scab diseases of citrus and its epidemiological implications. Phytopathology 86(10):1039
Timmer LW, Priest M, Broadbent P, Tan MK (1996) Morphological and pathological characterization of species of Elsinoe causing scab diseases of citrus. Phytopathology 86(10):1032
Tomlinson JA, Ostoja-Starzewska S, Webb K, Cole J, Barnes A, Dickinson M, Boonham N (2013) A loop-mediated isothermal amplification-based method for confirmation of Guignardia citricarpa in citrus black spot lesions. Eur J Plant Pathol 136(2):217–224. https://doi.org/10.1007/s10658-013-0168-9
Troncoso-Rojas R, Tiznado-Hernández ME (2014) Chapter 5—Alternaria alternata (Black Rot, Black Spot). In: Bautista-Baños S (ed) Postharvest decay. Academic Press, San Diego, pp 147–187
Van Gent-Pelzer MPE, Van Brouwershaven IR, Kox LFF, Bonants PJM (2007) A TaqMan PCR method for routine diagnosis of the quarantine fungus Guignardia citricarpa on citrus fruit. J Phytopathol 155(6):357–363. https://doi.org/10.1111/j.1439-0434.2007.01244.x
Vander Heyden Y, Nijhuis A, Smeyers-Verbeke J, Vandeginste BG, Massart DL (2001) Guidance for robustness/ruggedness tests in method validation. J Pharmaceut Biomed 24(5–6):723–753
Walker DM, Castlebury LA, Rossman AY, White JF Jr (2012) New molecular markers for fungal phylogenetics: two genes for species-level systematics in the Sordariomycetes (Ascomycota). Mol Phylogenet Evol 64(3):500–512. https://doi.org/10.1016/j.ympev.2012.05.005
Wang H, Qi M, Cutler AJ (1993) A simple method of preparing plant samples for PCR. Nucl Acids Res 21(17):4153–4154
Weiß CH (2007) StatSoft, Inc., Tulsa, OK: STATISTICA, version 8. AStA Adv Stat Anal 91(3):339–341. https://doi.org/10.1007/s10182-007-0038-x
White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols: a guide to methods and applications. Academic Press, New York, pp 315–322
Wikee S, Lombard L, Nakashima C, Motohashi K, Chukeatirote E, Cheewangkoon R, McKenzie EH, Hyde KD, Crous PW (2013) A phylogenetic re-evaluation of Phyllosticta (Botryosphaeriales). Stud Mycol 76(1):1–29. https://doi.org/10.3114/sim0019
Wulandari NF, To-anun C, Hyde KD, Duong LM, Jd G, Meffert JP, Groenewald JZ, Crous PW (2009) Phyllosticta citriasiana sp nov., the cause of Citrus tan spot of Citrus maxima in Asia. Fungal Divers 34:23–39
Yesuf M (2013) Pseudocercospora leaf and fruit spot disease of citrus: achievements and challenges in the citrus industry: a review. Agric Sci 4(07):324–328
Zavala MGM, Er HL, Goss EM, Wang NY, Dewdney M, van Bruggen AHC (2014) Genetic variation among Phyllosticta strains isolated from citrus in Florida that are pathogenic or nonpathogenic to citrus. Trop Plant Pathol 39:119–128
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.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Ethical approval
This article does not contain any studies with human participants or animals performed by any of the authors.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
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
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-020-10880-w