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