Integrative diagnosis of carrot cyst nematode (<Emphasis Type="Italic">Heterodera carotae</Emphasis>) using morphology and several molecular markers for an accurate identification |
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Authors: | Mehrdad Madani Juan E Palomares-Rius Nicola Vovlas Pablo Castillo Mario Tenuta |
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Institution: | 1.Department of Soil Science,University of Manitoba,Winnipeg,Canada;2.Instituto de Agricultura Sostenible (IAS), Consejo Superior de Investigaciones Científicas (CSIC),Campus de Excelencia Internacional Agroalimentario, (ceiA3),Córdoba,Spain;3.Istituto per la Protezione Sostenibile delle Piante (IPSP,Consiglio Nazionale delle Ricerche (CNR),Bari,Italy |
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Abstract: | 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. |
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