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Molecular diagnostics of the Mediterranean olive cyst nematode, Heterodera mediterranea Vovlas, Inserra & Stone, 1981 using conventional and real-time PCR

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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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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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Correspondence to Sergei A. Subbotin.

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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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