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Duplex real-time PCR assay for the simultaneous detection of Caliciopsis pinea and Fusarium circinatum in pine samples

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Abstract

Fusarium circinatum and Caliciopsis pinea are the causal agents of Pitch canker and Caliciopsis canker, respectively. These diseases affect pines and other conifers both in Europe and North America. The two pathogens cause similar bleeding cankers, especially at the early stage of colonization. Symptoms closely resembling those due to F. circinatum can be instead associated with C. pinea. Since F. circinatum is a quarantine organism, subjected to provisional emergency measures, its report immediately causes serious economic implications, while C. pinea, even if now emerging, is not regulated in the EU nor in the USA. For this reason, a reliable and accurate diagnostic tool able to distinguish between the two organisms was considered a priority. In this study, we developed and standardized a duplex real-time PCR assay allowing the simultaneous recognition of C. pinea and F. circinatum DNA in pine tissue in a reasonably short time and for amounts as small as 0.06 pg/μl. The molecular assay is, therefore, able to detect the infection even before symptoms have fully developed. The test was challenged with a very large set of strains (110 different isolates) collected in different regions of the world and host trees, and gave reliable results. The high efficiency of this method suggests its use as a standard diagnostic tool during phytosanitary controls. In addition, the duplex real-time PCR assay presented here is the first DNA-based method designed to detect C. pinea, which is becoming an increasing threat to pine stands both in North America and in Europe.

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References

  • Bland JM, Altman DG (1986) Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1(8476):307–310. https://doi.org/10.1016/S0140-6736(86)90837-8

    Article  PubMed  CAS  Google Scholar 

  • Bland JM, Altman DG (2007) Agreement between methods of measurement with multiple observations per individual. J Biopharm Stat 17:(4)571–582

  • Bonants P, te Witt R (2017) Molecular diagnostics in plant health. In: van Pelt-Verkuil E, van Leeuwen W, te Witt R (eds) Molecular diagnostics. Springer, Singapore, pp 175–202

    Chapter  Google Scholar 

  • Bordeaux JM, Lorenz WW, Dean JF. (2012) Biomarker genes highlight intraspecific and interspecific variations in the responses of Pinus taeda L. and Pinus radiata D. Don to Sirex noctilio F. acid gland secretions. Tree Physiol 2012 32 (10):1302–1312

  • Boutigny A-L, Scauflaire J, Ballois N, Ioos R (2017) Fusarium temperatum isolated from maize in France. Eur J Plant Path 148(4):997–1001. https://doi.org/10.1007/s10658-016-1137-x

    Article  Google Scholar 

  • Britz H, Coutinho TA, Wingfield MJ, Marasas WFO (2002) Validation of the description of Gibberella circinata and morphological differentiation of the anamorph Fusarium circinatum. Sydowia 54(1):9–22

  • Bragança H, Diogo E, Moniz F, Amaro P (2009) First report of pitch canker on pines caused by Fusarium circinatum. Plant Dis 93(10):1079. https://doi.org/10.1094/PDIS-93-10-1079A

    Article  Google Scholar 

  • Capretti P (1978) Caliciopsis pinea Peck parassita di Pinus pinaster e Pinus insignis. Phy Med 17(2):101–104

    Google Scholar 

  • Capretti P (1980) Pinus nigra var. austriaca, nuovo ospite di Caliciopsis pinea Peck. Inf Fitopatol 5:19–21

    Google Scholar 

  • Capretti P, Luchi N, Migliorini, D (2013) Occurrence of Caliciopsis pinea Peck on pine: interactions with other fungal pathogens. In: Proceedings of the IUFRO working party 7.02.02 Foliage, shoot and stem diseases of forest trees, ‘Biosecurity in natural forests and plantations, genomics and biotechnology for biosecurity in forestry’, Mendel University in Brno (Ed.), 20–25 May 2013, Brno, Czech Republic. p. 33

  • Carlucci A, Colatruglio L, Frisullo S (2007) First report of pitch canker caused by Fusarium circinatum on Pinus halepensis and P. pinea in Apulia (Southern Italy). Plant Dis 91(12):1683. https://doi.org/10.1094/PDIS-91-12-1683C

    Article  Google Scholar 

  • EPPO Reporting Service (2009) 2009/093 Situation of Gibberella circinata in France. http://archives.eppo.int/EPPOReporting/2009/Rse-0905.pdf

  • Fitzpatrick HM (1942) Revisionary studies in the Coryneliaceae. Mycologia 34:464–488

    Article  Google Scholar 

  • Gordon TR, Storer AJ, Wood DL (2001) The pitch canker epidemic in California. Plant Dis 85(11):1128–1139. https://doi.org/10.1094/PDIS.2001.85.11.1128

    Article  Google Scholar 

  • Harrison KJ (2009) Forest disease records on eastern white pine in Atlantic Canada: 1950 to 1996. Forestry Chron 85(4):604–608. https://doi.org/10.5558/tfc85604-4

    Article  Google Scholar 

  • Hepting GH, Roth ER (1946) Pitch canker, a new disease of some southern pines. J For 44:742–744

    Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Kubista M, Andrade JM, Bengtsson M, Forootan A, Jonák J, Lind K, Sindelka R, Sjöback R, Sjögreen B, Strömbom L, Ståhlberg A, Zoric N (2006) The real-time polymerase chain reaction. Mol Asp Med 27(2–3):95–125. https://doi.org/10.1016/j.mam.2005.12.007

    Article  CAS  Google Scholar 

  • Lamarche J, Potvin A, Pelletier G, Stewart D, Feau N, Alayon DI, Dale AL, Coelho A, Uzunovic A, Bilodeau GJ, Brière SC, Hamelin RC, Tanguay P (2015) Molecular detection of 10 of the most unwanted alien forest pathogens in Canada using real-time PCR. PLoS One 10(8):e0134265. https://doi.org/10.1371/journal.pone.0134265

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Landeras E, García P, Fernández Y, Braña M, Pérez-Sierra A, León M, Fernández-Alonso O, Méndez-Lodos S, Abad-Campos P, Berbegal M, Beltrán R, García-Jiménez J, Armengol J (2005) Outbreak of pitch canker caused by Fusarium circinatum on Pinus spp. in northern Spain. Plant Dis 89(9):1015. https://doi.org/10.1094/PD-89-1015A

    Article  Google Scholar 

  • Lanier PL (1965) Note sur une espéce cryptogamique nouvelle pour la France sur différents pins dont le pin maritime des Landes. Rev For Française 1:34–41

    Article  Google Scholar 

  • Migliorini D, Ghelardini L, Tondini E, Luchi N, Santini A (2015) The potential of symptomless potted plants for carrying invasive soil-borne plant pathogens. Divers Distrib 21:1218–1229

  • Munck I, Livingston W, Lombard K, Luther T, Ostrofsky W, Weimer J, Wyka S, Broders K (2015) Extent and severity of Caliciopsis canker in New England, USA: an emerging disease of eastern white pine (Pinus strobus L.). Forests 6(11):4360–4373. https://doi.org/10.3390/f6114360

    Article  Google Scholar 

  • Munck I, Luther T, Wyka S, Keirstead D, McCracken K, Ostrofsky W, Searles W, Lombard K, Allen B (2016) Soil and stocking effects on Caliciopsis canker of Pinus strobus L. Forests 7(11):269. https://doi.org/10.3390/f7110269

    Article  Google Scholar 

  • Ramsfield TD, Dobbie K, Dick MA, Ball RD (2008) Polymerase chain reaction-based detection of Fusarium circinatum, the causal agent of pitch canker disease. Mol Ecol Res 8(6):1270–1273. https://doi.org/10.1111/j.1755-0998.2008.02188.x

    Article  CAS  Google Scholar 

  • Ray WW (1936) Pathogenicity and cultural experiments with Caliciopsis pinea. Mycologia 28:201–208

    Article  Google Scholar 

  • Rehm H (1896) Die Pilze Deutschlands, Oesterreichs und der Schweiz. III. Abtheilung: Ascomyceten: Hysteriaceen und Discomyceten. In: Richter PG (ed) Dr. L. Rabenhorst’s Kryptogamen-Flora von Deutschland, Oesterreich und der Schweiz. Eduard Kummer, Zweite Auflage, Leipzig. pp 382–414

  • Santini A, Ghelardini L, De Pace C, Desprez-Loustau ML, Capretti P, Chandelier A, Cech T, Chira D, Diamandis S, Gaitniekis T, Hantula J, Holdenrieder O, Jankovsky L, Jung T, Jurc D, Kirisits T, Kunca A, Lygis V, Malecka M, Marcais B, Schmitz S, Schumacher J, Solheim H, Solla A, Szabo` I, Tsopelas P, Vannini A, Vettraino AM, Webber J, Woodward S, Stenlid J (2013) Biogeographical patterns and determinants of invasion by forest pathogens in Europe. New Phytol 197(1):238–250. https://doi.org/10.1111/j.1469-8137.2012.04364.x

    Article  PubMed  CAS  Google Scholar 

  • Stanosz GR, Blodgett JT, Smith DR, Kruger EL (2001) Water stress and Sphaeropsis sapinea as a latent pathogen of red pine seedlings. New Phytol 149(3):531–538. https://doi.org/10.1046/j.1469-8137.2001.00052.x

    Article  Google Scholar 

  • Wingfield MJ, Hammerbacher A, Ganley RJ, Steenkamp ET, Gordon TR, Wingfield BD, Coutinho TA (2008) Pitch canker caused by Fusarium circinatum—a growing threat to pine plantations and forests worldwide. Australas Plant Pathol 37(4):319–334. https://doi.org/10.1071/AP08036

    Article  Google Scholar 

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Acknowledgments

The authors wish to thank Dr. I. Munck (U.S. Department of Agriculture (USDA) Forest Service), Prof. K.D. Broders (Colorado State University), Dr. Isaac Garrido-Benavent (Grupo de Ecología Microbiana y Geomicrobiología Museo Nacional de Ciencias Naturales, Madrid, Spain), Prof. Laura Mugnai and Prof. P. Capretti (DISPAA, University of Florence, Italy), Prof. Glen R. Stanosz (University of Wisconsin, USA), Prof. Julio J. Diez-Castro (Universidad de Valladolid, Spain), and Dr. Alan R. Wood (Weed Division, ARC-Plant Protection Research Institute, Stellenbosch, South Africa) for having generously provided fungal isolates; Dr. Francesco Pecori (IPSP-CNR, Italy) for lab assistance; and Dr. Trudy Paap (FABI, University of Pretoria, South Africa) for the critical review and for English language editing of the manuscript. This work has been developed in the frame of the EU-COST Action FP 1406 “Pine pitch canker - strategies for management of Gibberella circinata in greenhouses and forests” (PINESTRENGTH).

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Authors and Affiliations

  1. National Research Council (IPSP-CNR), Institute for Sustainable Plant Protection, Via Madonna del Piano, 10 I-50019 Sesto Fiorentino, Florence, Italy

    Nicola Luchi, Alessia Lucia Pepori, Paola Bartolini & Alberto Santini

  2. ANSES Laboratoire de la Santé des Végétaux, unité de mycologie, Domaine de Pixérécourt Bât. E, F-54220, Malzéville, France

    Renaud Ioos

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  1. Nicola Luchi
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  2. Alessia Lucia Pepori
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  3. Paola Bartolini
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  4. Renaud Ioos
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  5. Alberto Santini
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Correspondence to Nicola Luchi.

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This study does not contain any studies with human participants or animals performed by any of the authors.

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Luchi, N., Pepori, A.L., Bartolini, P. et al. Duplex real-time PCR assay for the simultaneous detection of Caliciopsis pinea and Fusarium circinatum in pine samples. Appl Microbiol Biotechnol 102, 7135–7146 (2018). https://doi.org/10.1007/s00253-018-9184-1

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