Skip to main content

Advertisement

Log in

Development of a quantitative PCR assay for the detection of Piggotia coryli, the causal agent of hazelnut anthracnose

  • Original Article
  • Published:
Journal of Plant Pathology Aims and scope Submit manuscript

Abstract

A novel real-time PCR assay was developed for the detection of Piggotia coryli, the causal agent of hazelnut anthracnose. A molecular tool for sensitive detection and quantification of P. coryli in symptomatic and asymptomatic host tissues was required to better understand P. coryli biology and epidemiology. Species-specific primers for P. coryli were designed based on the sequence of the β-tubulin gene. The amplification efficiency of pure target DNA was 93.2%, wet lab-tested limit of detection (LOD) was fixed at 8 pg/PCR reaction with maximum values of repeatability and reproducibility. In samples collected from infected orchards, the assay consistently revealed the presence of the pathogen in all symptomatic specimens, as well as in some asymptomatic tissues. This molecular tool will be of substantial aid in detecting and quantifying P. coryli, even in latent state and in different hazelnut tissues.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

Data availability

The data that support the findings of this study are available from the corresponding upon request.

References

  • Anderson JP, Gleason CA, Foley RC, Thrall PH, Burdon JB, Singh KB (2010) Plants versus pathogens: an evolutionary arms race. Funct Plant Biol 37(6):499–512. https://doi.org/10.1071/FP09304. PMID: 21743794; PMCID: PMC3131095

    Article  PubMed  PubMed Central  Google Scholar 

  • Arbefeville S, Harris A, Ferrieri P (2017) Comparison of sequencing the D2 region of the large subunit ribosomal RNA gene (MicroSEQ®) versus the internal transcribed spacer (ITS) regions using two public databases for identification of common and uncommon clinically relevant fungal species. J Microbiol Methods 140:40–46

    Article  CAS  PubMed  Google Scholar 

  • 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 Technol 37(2):115–126. https://doi.org/10.1016/j.tifs.2014.03.008

    Article  CAS  Google Scholar 

  • Bustin SA, Benes V, Garson JA et al (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622. https://doi.org/10.1373/clinchem.2008.112797

    Article  CAS  PubMed  Google Scholar 

  • Calderon C, Ward E, Freeman J, McCartney A (2002) Detection of airborne fungal spores sampled by rotating-arm and Hirst-type spore traps using polymerase chain reaction assays. J Aerosol Sci 33:283–296

    Article  CAS  Google Scholar 

  • Druzhinina IS, Kopchinskiy AG, Kubicek CP (2006) The first 100 Trichoderma species characterized by molecular data. Mycoscience 47:55

    Article  CAS  Google Scholar 

  • Einax E, Voigt K (2003) Oligonucleotide primers for the universal amplification of β-tubulin genes facilitate phylogenetic analyses in the regnum Fungi. Org Divers Evol 3:185–194. https://doi.org/10.1078/1439-6092-00069

    Article  Google Scholar 

  • El-Sayed A, Kamel M (2020) Climatic changes and their role in emergence and re-emergence of diseases. Environ Sci Pollut Res 27:22336–22352. https://doi.org/10.1007/s11356-020-08896-w

    Article  CAS  Google Scholar 

  • Falacy JS, Grove GG, Mahaffee WF et al (2007) Detection of Erysiphe necator in air samples using the polymerase chain reaction and species-specific primers. Phytopathology 97:1290–1297

    Article  CAS  PubMed  Google Scholar 

  • Fraaije BA, Cools HJ, Fountaine J et al (2005) Role of ascospores in further spread of QoI-resistant cytochrome b alleles (G143A) in field populations of Mycosphaerella graminicola. Phytopathology 95:933–941

    Article  CAS  PubMed  Google Scholar 

  • Gervasi F, Scortichini M (2009) Detection of Pseudomonas avellanae from hazelnut twigs by TaqMan real-time PCR. J Plant Pathol 573–578

  • Gianetti G, Cravero S, Morone C (1994) Further observations about bioepidemiology of Monostichella coryli (Deuteromycetes, Melanconiales), causal organism of bud-rot of hazel. Acta Horticulturae (Netherlands)

  • Hughes KJ, Tomlinson JA, Giltrap PM et al (2011) Development of a real-time PCR assay for detection of Phytophthora kernoviae and comparison of this method with a conventional culturing technique. Eur J Plant Pathol 131:695–703

    Article  CAS  Google Scholar 

  • ISTAT (2021) Available at: https://www.istat.it. Accessed 10 Oct 2021

  • Kumar S, Stecher G, Li M et al (2018) MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35:1547–1549. https://doi.org/10.1093/molbev/msy096

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Luo Y, Lichtemberg PS, Niederholzer FJ et al (2019) Understanding the process of latent infection of canker-causing pathogens in stone fruit and nut crops in California. Plant Dis 103:2374–2384

    Article  CAS  PubMed  Google Scholar 

  • Mancini V, Murolo S, Romanazzi G (2016) Diagnostic methods for detecting fungal pathogens on vegetable seeds. Plant Pathol 65:691–703. https://doi.org/10.1111/ppa.12515

    Article  CAS  Google Scholar 

  • Martinelli F, Scalenghe R, Davino S et al (2015) Advanced methods of plant disease detection. A review. Agron Sustain Dev 35:1–25. https://doi.org/10.1007/s13593-014-0246-1

    Article  Google Scholar 

  • Mazzaglia A, Drais MI, Turco S et al (2021) First report of Erysiphe corylacearum causing powdery mildew on Corylus avellana in Spain. New Dise Rep

  • Merkes CM, Klymus KE, Allison MJ et al (2019) Generic qPCR Limit of Detection (LOD)/Limit of Quantification (LOQ) calculator. R Script. US Geol Surv Code Release

  • Migliorini D, Ghelardini L, Tondini E et al (2015) The potential of symptomless potted plants for carrying invasive soilborne plant pathogens. Divers Distrib 21:1218–1229

    Article  Google Scholar 

  • Molnar TJ, Walsh E, Capik JM et al (2013) A real-time PCR assay for early detection of eastern filbert blight. Plant Dis 97:813–818

    Article  CAS  PubMed  Google Scholar 

  • O’Donnell K, Ward TJ, Geiser DM et al (2004) Genealogical concordance between the mating type locus and seven other nuclear genes supports formal recognition of nine phylogenetically distinct species within the Fusarium graminearum clade. Fungal Genet Biol 41:600–623

    Article  PubMed  Google Scholar 

  • Pesante A (1966) Disseccamento dei rametti di Nocciòlo causato da un Gloeosporium. Boll. Lab. sper. Oss Fitopatol NS 27:17–21

    Google Scholar 

  • Pilotti M, Tizzani L, Brunetti A et al (2010) Molecular identification of Fomitiporia mediterranea on declining and decayed hazelnut. J Plant Pathol 115–129

  • Pothier JF, Pagani MC, Pelludat C et al (2011) A duplex-PCR method for species-and pathovar-level identification and detection of the quarantine plant pathogen Xanthomonas arboricola pv. pruni. J Microbiol Methods 86:16–24

    Article  CAS  PubMed  Google Scholar 

  • Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132:365–386. https://doi.org/10.1385/1-59259-192-2:365. PMID: 10547847

  • Sabelis MW, Bruin J (1996) 1.5.3. Evolutionary ecology: Life history patterns, food plant choice and dispersal. In: Lindquist EE, Sabelis MW, Bruin J (eds) World Crop Pests, Elsevier, Volume 6, pp 329–366. ISSN: 1572-4379; ISBN: 9780444886286. https://doi.org/10.1016/S1572-4379(96)80020-0

  • Scortichini M, Marchesi U (2001) Sensitive and specific detection of Pseudomonas avellanae using primers based on 16S rRNA gene sequences. J Phytopathol 149:527–532

    Article  CAS  Google Scholar 

  • Turco S, Bastianelli G, Morales-Rodrìguez C et al (2021) Development of a TaqMan qPCR assay for the detection and quantification of Gnomoniopsis castaneae in chestnut tissues. For Pathol e12701

  • Valasek MA, Repa JJ (2005) The power of real-time PCR. Adv Physiol Educ 29:151–159

    Article  PubMed  Google Scholar 

  • West JS, Kimber RBE (2015) Innovations in air sampling to detect plant pathogens. Ann Appl Biol 166:4–17

    Article  PubMed  PubMed Central  Google Scholar 

  • Ye J, Coulouris G, Zaretskaya I et al (2012) Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction. BMC Bioinform 13:134. https://doi.org/10.1186/1471-2105-13-134

    Article  CAS  Google Scholar 

Download references

Funding

This work has been supported by the European Commission under the Grant Agreement number 774571 (project PANTHEON ‘Precision farming of hazelnut orchards’).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Angelo Mazzaglia.

Ethics declarations

Competing interests

The authors have no relevant financial or non-financial interests to disclose.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

42161_2023_1326_MOESM1_ESM.docx

Supplementary file1 Melting curve analysis from the real time PCR assay, the peaks at 85.8 °C correspond to the dissociation curve of four specific amplification products obtained (DOCX 35 KB)

Supplementary file2 List of primers used for the molecular characterization of Piggotia coryli strains (DOCX 20 KB)

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Drais, M.I., Turco, S., D’Attilia, C. et al. Development of a quantitative PCR assay for the detection of Piggotia coryli, the causal agent of hazelnut anthracnose. J Plant Pathol 105, 507–516 (2023). https://doi.org/10.1007/s42161-023-01326-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s42161-023-01326-z

Keywords

Navigation