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Título: | Integrative diagnosis of carrot cyst nematode (Heterodera carotae) using morphology and several molecular markers for an accurate identification |
Autor: | Madani, Mehrdad; Palomares Rius, Juan E. CSIC ORCID; Vovlas, Nicola; Castillo, Pablo CSIC ORCID ; Tenuta, Mario | Palabras clave: | Molecular identification PCR Real time PCR Species-specific primer |
Fecha de publicación: | abr-2018 | Editor: | Springer Nature | Citación: | European Journal of Plant Pathology 150(4): 1023-1039 (2018) | Resumen: | 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. | Versión del editor: | https://doi.org/10.1007/s10658-017-1342-2 | URI: | http://hdl.handle.net/10261/180166 | DOI: | 10.1007/s10658-017-1342-2 | ISSN: | 0929-1873 | E-ISSN: | 1573-8469 |
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