Abstract
The Mediterranean olive cyst nematode, Heterodera mediterranea, is a pest of olive trees that is found in Spain, Italy, and Tunisia. In this paper, conventional and real-time PCR assays for the identification of H. mediterranea was developed. The hsp90 gene fragment was used to design a species-specific primer and a TaqMan probe. The specificity of the primer/probe set was tested in singleplex or multiplex reactions against Mediterranean olive cyst nematode and non-target nematodes. In multiplex real-time PCR experiments with the specific and universal primer/probe sets, fluorescent signals were simultaneously monitored for hsp90 and D3 of 28S rRNA target genes. The results of the experiments showed that the assays with species-specific primer/probe were sensitive enough to detect the DNA from 0.03 of a second-stage juvenile (J2) H. mediterranea in a reaction tube.
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References
Archidona-Yuste, A., Wiegand, T., Castillo, P., & Navas-Cortés, J. A. (2020). Spatial structure and soil properties shape local community structure of plant-parasitic nematodes in cultivated olive trees in southern Spain. Agriculture, Ecosystems & Environment, 287, 106688. https://doi.org/10.1016/j.agee.2019.106688
Castillo, P., Vovlas, N., Nico, A. I., & Jiménez-Diaz, R. M. (1999). Infection of olive trees by Heterodera mediterranea in orchards in southern Spain. Plant Disease, 83, 710–713. https://doi.org/10.1094/PDIS.1999.83.8.710
Castillo, P., Nico, A. I., Navas-Cortés, J. A., Landa, B. B., Jiménez-Díaz, R. M., & Vovlas, N. (2010). Plant-parasitic nematodes attacking olive trees and their management. Plant Disease, 94, 145–280. https://doi.org/10.1094/PDIS-94-2-0148
Chenna, R., Sugawara, H., Koike, T., Lopez, R., Gibson, T. J., Higgins, D. G., & Thompson, J. D. (2003). Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Research, 31, 3497–3500. https://doi.org/10.1093/nar/gkg500
Coolen, W. A. (1979). Methods for the extraction of Meloidogyne spp. and other nematodes from roots and soil. In F. Lamberti & C. E. Taylor (Eds.), Root-knot nematodes (Meloidogyne species) systematics (pp. 319–329). Biology and Control. Academic Press.
Guesmi-Mzoughi, I., Troccoli, A., Fanelli, E., Radicci, V., Regaieg, H., Hadj-Naser, F., Horrigue-Raouani, N., & De Luca, F. (2018). First report of the cyst nematode Heterodera mediterranea on olive trees in Tunisia. Tunisian Journal of Plant Protection, 13, 69–77.
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. https://doi.org/10.1111/j.1439-0434.2004.00835.x
Powers, T., Skantar, A., Harris, T., Higgins, R., Mullin, P., Hafez, S., Handoo, Z., Todd, T., & Powers, K. (2019). DNA barcoding evidence for the north American presence of alfalfa cyst nematode, Heterodera medicaginis. Journal of Nematology, 51, 1–17. https://doi.org/10.21307/jofnem-2019-016
Roubtsova, T. V., & Subbotin, S. A. (2022). Molecular diagnostics of the pigeon pea cyst nematode, Heterodera cajani Koshy, 1967, using real-time PCR. Nematology, 24, 85–90. https://doi.org/10.1163/15685411-bja10113
Sabo, A., Vovlas, N., & Ferris, V. R. (2001). Phylogenetic relationships based on ribosomal DNA data for four species of cyst nematodes from Italy and one from Syria. Journal of Nematology, 33, 183–190.
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.
Skantar, A. M., Handoo, Z. A., Zanakis, G. N., & Tzortzakakis, E. A. (2012). Molecular and morphological characterization of the corn cyst nematode, Heterodera zeae, from Greece. Journal of Nematology, 44, 58–66.
Vovlas, N., & Inserra, R. N. (1983). Biology of Heterodera mediterranea. Journal of Nematology, 15, 571–576.
Vovlas, N., Inserra, R. N., & Stone, A. R. (1981). Heterodera mediterranea n. sp. (Nematoda: Heteroderidae) on Pistacia lentiscus in southern Italy. Nematologica, 27, 129–138. https://doi.org/10.1163/187529281X00188
Wang, X., Bergstrom, G. C., Chen, S., Thurston, D. M., Cummings, J. A., Handoo, Z. A., Hult, M. N., & Skantar, A. M. (2017). First report of the soybean cyst nematode, Heterodera glycines, in New York. Plant Disease, 101, 1957. https://doi.org/10.1094/PDIS-06-17-0803-PDN
Yoder, M., De Ley, I. T., King, I. W., Mundo-Ocampo, M., Mann, J., Blaxter, M., Poiras, L., & De Ley, P. (2006). DESS: A versatile solution for preserving morphology and extractable DNA of nematodes. Nematology, 8, 367–376. https://doi.org/10.1163/156854106778493448
Acknowledgements
The authors thank Drs V.N. Chizhov, I.H. Elekçioğlu, G. Karssen, D. Mollov, R. Robbins, J. Rowe, N. Vovlas and W. Ye for providing cyst nematode samples. This work was sponsored by the FarmBill USDA grant: ‘Enhancing diagnostics of cyst forming nematodes of the genus Heterodera’, Cooperative Agreement Number: AP20PPQS&T00C078/ 20-0256-000-FR.
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Subbotin, S.A., Burbridge, J., Palomares-Rius, J.E. et al. Molecular diagnostics of the Mediterranean olive cyst nematode, Heterodera mediterranea Vovlas, Inserra & Stone, 1981 using conventional and real-time PCR. Eur J Plant Pathol 165, 179–187 (2023). https://doi.org/10.1007/s10658-022-02598-4
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DOI: https://doi.org/10.1007/s10658-022-02598-4