European Journal of Plant Pathology

, Volume 150, Issue 4, pp 1023–1039 | Cite as

Integrative diagnosis of carrot cyst nematode (Heterodera carotae) using morphology and several molecular markers for an accurate identification

  • Mehrdad Madani
  • Juan E. Palomares-Rius
  • Nicola Vovlas
  • Pablo CastilloEmail author
  • Mario Tenuta


Cyst nematodes obtained from commercial carrot fields in Ontario (Canada) and northern and southern Italy were subjected to morphological and molecular examination. Morphology of cyst cone tops, males and second-stage juveniles (J2) indicated the nematode species was the Carrot Cyst Nematode (CaCN), Heterodera carotae. The sequence of the Internal Transcribed Spacer (ITS), D2-D3 region of the 28S gene of ribosomal RNA, cytochrome oxidase I of mitochondrial DNA (coxI), and a heat shock protein gene (hsp90), from single cysts were also examined. Sequences of ITS and D2-D3 placed all the nematodes with Heterodera carotae and other Heterodera spp. belonging to the Goettingiana group in the same clade. The novel nine coxI sequences obtained also clustered in a well-supported phylogenetic clade for H. carotae. Similarly, the six new hsp90 sequences of H. carotae generated in this study were placed in a well-supported clade (PP = 1.00) together with other two sequences of H. carotae from Greece. Restriction Fragment Length Polymorphism (RFLP) of ITS-PCR products gave a restriction pattern for RsaI different than H. carotae but the other 6 restriction patterns were similar as described in former research. A diagnostic conventional PCR method was developed based on a primer set to be specific for H. carotae using coxI sequence. These primers were also used in real time PCR to generate a melt curve specific to H. carotae. Limit of detection for CaCN in conventional PCR reaction was a single J2.


Molecular identification PCR real time PCR species-specific primer 



The authors thank Dr. Mary Ruth McDonald for providing soil and carrot samples from Canada and C. Cantalapiedra-Navarrete (IAS-CSIC) for her excellent technical assistance in the laboratory. This research was supported by grant AGR-136 “Consejería de Economía, Innvovación y Ciencia” from Junta de Andalucía and “Una manera de hacer Europa” from Unión Europea, Fondo Europeo de Desarrollo regional, and the Canada Research Chair Program to M. Tenuta. Juan E. Palomares-Rius was a recipient of a “Juan de La Cierva” contract from Ministerio de Economía y Competitividad of Spain.

Compliance with ethical standards

Conflict of interest

All the authors certify that 1) do not have any actual or potential conflict of interest, 2) the study described is original and has not been published previously, and is not under consideration for publication elsewhere, 3) all prevailing local, national and international regulations and conventions, and normal scientific ethical practices, have been respected. We also certify that all authors have reviewed the manuscript and approved the final version of manuscript before submission.

Research involving human participants and/or animals

No specific permits were required for the described fieldwork studies. Permission for sampling was granted by the landowner.

Informed consent

All the authors certify that the work carried out in this research followed the principles of ethical and professional conduct have been followed. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


  1. Ambrogioni, L. (1971). Osservazioni morfologiche e biologiche su Heterodera carotae Jones 1950. Redia, 52, 241–258.Google Scholar
  2. Amiri, S., Subbotin, S. A., & Moens, M. (2001). An efficient method for identification of the Heterodera schachtii sensu stricto group using PCR with specific primers. Nematologia Mediterranea, 29, 241–246.Google Scholar
  3. Amiri, S., Subbotin, S. A., & Moens, M. (2002). Identification of the beet cyst nematode Heterodera schachtii by PCR. European Journal of Plant Pathology, 108, 497–506.CrossRefGoogle Scholar
  4. Berney, M. F., & Bird, G. W. (1992). Distribution of Heterodera carotae and Meloidogyne hapla in Michigan carrot production. Journal of Nematology, 24, 776–778.PubMedPubMedCentralGoogle Scholar
  5. Berry, O., & Sarre, S. D. (2007). Gel-free species identification using melt-curve analysis. Molecular Ecology Notes, 7, 1–4.CrossRefGoogle Scholar
  6. Castresana, J. (2000). Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Molecular Biology and Evolution, 17, 540–552.CrossRefPubMedGoogle Scholar
  7. Correia, F. J. S., Abrantes, I. M., & De, O. (1997). An improved technique for mounting Heterodera cysts for light microscopy. Nematologica, 43, 507–509.CrossRefGoogle Scholar
  8. Darriba, D., Taboada, G. L., Doallo, R., & Posada, D. (2012). jModelTest 2: more models, new heuristics and parallel computing. Nature Methods, 9, 772.CrossRefPubMedPubMedCentralGoogle Scholar
  9. De Ley, P., Félix, M. A., Frisse, L. M., Nadler, S. A., Sternberg, P. W., & Thomas, K. W. (1999). Molecular and morphological characterisation of two reproductively isolated species with mirror-image anatomy (Nematoda: Cephalobidae). Nematology, 1, 591–612.CrossRefGoogle Scholar
  10. De Luca, F., Vovlas, N., Lucarelli, G., Troccoli, A., Radicci, V., Fanelli, E., Cantalapiedra-Navarrete, C., Palomares-Rius, J. E., & Castillo, P. (2013). Heterodera elachista the Japanese cyst nematode parasitizing corn in Northern Italy: integrative diagnosis and bionomics. European Journal of Plant Pathology, 136, 857–872.CrossRefGoogle Scholar
  11. Derycke, S., Remerie, T., Vierstraete, A., Backeljau, T., Vanfleteren, J., Vincx, M., & Moens, T. (2005). Mitochondrial DNA variation and cryptic speciation within the free-living marine nematode Pellioditis marina. Marine Ecology Progress Series, 300, 91–103.CrossRefGoogle Scholar
  12. Derycke, S., Rigaux, V. J., Backeljau, A., & Moens, T. (2010). Exploring the use of cytochrome oxidase c subunit 1 (COI) for DNA barcoding of free-living marine nematodes. PloS One, 5, e13716.CrossRefPubMedPubMedCentralGoogle Scholar
  13. Greco, N., D’Addabbo, T., Brandonisio, A., & Elia, F. (1994). Damage to Italian crops caused by cyst-forming nematodes. Journal of Nematology, 25, 836–842.Google Scholar
  14. Hall, T. A. (1999). BioEdit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/NT. Nucleic Acids Symposium Series, 41, 95–98.Google Scholar
  15. Hebert, P. D. N., Cywinska, A., Ball, S. L., & DeWaard, J. R. (2003). Biological identifications through DNA barcodes. Proceedings of the Royal Society of London B Biological Sciences, 270, 313–321.CrossRefGoogle Scholar
  16. Jones, F. G. W. (1950). A new species of roots eelworm attacking carrots. Nature, 165, 81.CrossRefGoogle Scholar
  17. Joyce, S. A., Burnell, A. M., & Powers, T. O. (1994). Characterization of Heterorhabditis isolates by PCR amplification of segments of mtDNA and rDNA genes. Journal of Nematology, 26, 260–270.PubMedPubMedCentralGoogle Scholar
  18. Katoh, K., & Standley, D. M. (2013). MAFFT multiple sequence alignment 542 software version 7: improvements in performance and usability. Molecular Biology and Evolution, 30, 772–780.CrossRefPubMedPubMedCentralGoogle Scholar
  19. Maafi, Z., Subbotin, S. A., & Moens, M. (2003). Molecular identification of cyst-forming nematodes (Heteroderidae) from Iran and a phylogeny based on ITS-rDNA sequences. Nematology, 5, 99–111.CrossRefGoogle Scholar
  20. Madani, M., Vovlas, N., Castillo, P., Subbotin, S. A., & Moens, M. (2004). Molecular characterization of cyst nematode species (Heterodera spp.) from the Mediterranean Basin using RFLPs and sequences of ITS-rDNA. Journal of Phytopathology, 152, 229–234.CrossRefGoogle Scholar
  21. Mathews, H.J.P. (1975). Heterodera carotae. CIH Descriptions of plant parasitic nematodes. Set 5, No. 61 St Albans, UK, Commonwealth Institute of Helminthology. 1-4.Google Scholar
  22. Nicholas, K. B., & Nicholas, H. B. Jr. (1997). GenDoc: a tool for editing and annotating multiple sequence alignment. Distributed by the authors (
  23. Ou, S., Peng, D., Liu, X., & Moens, M. (2008). Identification of Heterodera glycines using PCR with sequence characterised amplified region (SCAR) primers. Nematology, 10, 397–403.CrossRefGoogle Scholar
  24. Page, R. D. (1996). TreeView: an application to display phylogenetic trees on personal computers. Computer Applications in the Biosciences, 12, 357–358.PubMedGoogle Scholar
  25. Ronquist, F., & Huelsenbeck, J. P. (2003). MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics, 19, 1572–1574.CrossRefPubMedGoogle Scholar
  26. Seinhorst, J. W. (1966). Killing nematodes for taxonomic study with hot F.A. 4: 1. Nematologica, 12, 178.CrossRefGoogle Scholar
  27. Skantar, A. M., & Carta, L. K. (2004). Molecular characterization and phylogenetic evaluation of the hsp90 gene from selected nematodes. Journal of Nematology, 36, 466–480.PubMedPubMedCentralGoogle Scholar
  28. Skantar, A. M., & Carta, L. K. (2005). Phylogenetic evaluation of nucleotide and protein sequences from the heat shock protein 90 gene of selected nematodes. Journal of Nematology, 36, 466–480.Google Scholar
  29. Skantar, A. M., Handoo, Z. A., Carta, L. K., & Chitwood, D. J. (2007). Morphological and molecular identification of Globodera pallida associated with potato in Idaho. Journal of Nematology, 39, 133–144.PubMedPubMedCentralGoogle Scholar
  30. Soliman, R. H., & Othman, A. A. (2009). Evaluation of DNA Melting Curve Analysis Real-Time PCR for Detection and Differentiation of Cryptosporidium Species. Parasitologists United Journal (PUJ), 2, 47–54.Google Scholar
  31. Subbotin, S. A. (2015). Heterodera sturhani sp. n. from China, a new species of the Heterodera avenae species complex (Tylenchida: Heteroderidae). Russian Journal of Nematology, 23, 145–152.Google Scholar
  32. Subbotin, S. A., Waeyenberge, L., & Moens, M. (2000). Identification of cyst forming nematodes of the genus Heterodera (Nematoda: Heteroderidae) based on the ribosomal DNA-RFLP. Nematology, 2, 153–164.CrossRefGoogle Scholar
  33. Subbotin, S. A., Vierstraete, A., De Ley, P., Rowe, J., Waeyenberge, L., Moens, M., & Vanfleteren, J. R. (2001). Phylogenetic relationships within the cyst-forming nematodes (Nematoda, Heteroderidae) based on analysis of sequences from the ITS regions of ribosomal DNA. Molecular Phylogenetics and Evolution, 21, 1–16.CrossRefPubMedGoogle Scholar
  34. Subbotin, S. A., Sturhan, D., Rumpenhorst, H. J., & Moens, M. (2003). Molecular and morphological characterisation of the Heterodera avenae species complex (Tylenchida: Heteroderidae). Nematology, 5, 515–518.CrossRefGoogle Scholar
  35. Subbotin, S. A., Mundo-Ocampo, M., & Baldwin, J. G. (2010). Systematics of cyst nematodes (Nematoda: Heteroderinae). In D. J. Hunt & R. N. Perry (Eds.), Nematology Monographs and Perspectives 8B. The Netherlands: Leiden 512 pp.Google Scholar
  36. Thompson, J., Gibson, T., Plewniak, F., Jeanmougin, F., & Higgins, D. (1997). The CLUSTAL X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research, 25, 4876–4882.CrossRefPubMedPubMedCentralGoogle Scholar
  37. Toumi, F., Waeyenberge, L., Viaene, N., Dababat, A., Nicol, J. M., Ogbonnaya, F., & Moens, M. (2013). Development of two species-specific primer sets to detect the cereal cyst nematodes Heterodera avenae and Heterodera filipjevi. European Journal of Plant Pathology, 136, 613–624.CrossRefGoogle Scholar
  38. Varga, A., & James, D. (2006). Real-time RT-PCR and SYBR Green I melting curve analysis for the identification of Plum pox virus strains C, EA, and W: Effect of amplicon size, melt rate, and dye translocation. Journal of Virological Methods, 132, 146–153.CrossRefPubMedGoogle Scholar
  39. Vovlas, N., Vovlas, A., Leonetti, P., Liébanas, G., Castillo, P., Subbotin, S., & Palomares-Rius, J. E. (2015). Parasitism effects on White clover by root-knot and cyst nematodes and molecular separation of Heterodera daverti from H. trifolii. European Journal of Plant Pathology, 143, 833–845.CrossRefGoogle Scholar
  40. Waeyenberge, L., Viaene, N., Subbotin, S. A., & Moens, M. (2009). Molecular identification of Heterodera spp., an overview of fifteen years of research. Proceedings of the first workshop of the international cereal cyst nematode initiative, 21–23 October 2009 (pp. 109–114). Antalya: CIMMYT.Google Scholar
  41. Wouts, W. M., & Weischer, B. (1977). Eine klassifizierung von fünfzehn in Westeuropa häufigen artern der Heteroderinae auf grund von larvenmerkmalen. Nematologica, 23, 289–310.CrossRefGoogle Scholar
  42. Yu, Q., Ponomareva, E., Van Dyk, D., McDonald, M. R., Sun, F., Madani, M., & Tenuta, M. (2017). First report of the carrot cyst nematode (Heterodera carotae Jones) from carrot fields in Ontario, Canada. Plant Disease, 101, 1056–1056.Google Scholar

Copyright information

© Koninklijke Nederlandse Planteziektenkundige Vereniging 2017

Authors and Affiliations

  • Mehrdad Madani
    • 1
  • Juan E. Palomares-Rius
    • 2
  • Nicola Vovlas
    • 3
  • Pablo Castillo
    • 2
    Email author
  • Mario Tenuta
    • 1
  1. 1.Department of Soil ScienceUniversity of ManitobaWinnipegCanada
  2. 2.Instituto de Agricultura Sostenible (IAS), Consejo Superior de Investigaciones Científicas (CSIC)Campus de Excelencia Internacional Agroalimentario, (ceiA3)CórdobaSpain
  3. 3.Istituto per la Protezione Sostenibile delle Piante (IPSPConsiglio Nazionale delle Ricerche (CNR)BariItaly

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