Considering the growing trade of seed potato, reliable diagnostic protocols are required for the detection of regulated nematode species. In this study, a specific and sensitive multiplex Taqman-based real-time PCR method was developed in order to detect and identify Globodera pallida, G. rostochiensis and Heterodera schachtii. The newly designed primers and probes enabled the detection of all the target populations tested and with no cross-reaction for closely related non-target species (55 populations tested). The limit of detection (LOD) was one juvenile for G. rostochiensis and G. pallida and five juveniles for H. schachtii. For monitoring potato cyst nematodes, this analytical tool would extend the number of cyst investigated as five juveniles can be detected among 50 cysts in a sample. Furthermore, this multiplex assay detects DNA of the three targeted species in template DNA obtained directly from float material after nematode extraction from soil.

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Peruvian potato cyst nematode populations were analysed to assess both their inter- and intraspecific similarities. ITS--RFLP and two satellite DNA sequences were used as taxonomic tools. Both techniques have confirmed that the Peruvian populations have as their closest relatives the European Globodera pallida, despite the detection of clear differences that prevents us from assigning these South American populations unambiguously to any Globodera species. A more precise study of the variability of these Peruvian populations was investigated and they were compared with the imported European populations using protein (2-DGE) and DNA (RAPD) datasets. The clear distinction between the Peruvian and the European populations was confirmed and, inside each group, no correlation was found between the pathotype classification and the observed clustering of the populations. Surprisingly, while RAPDs revealed a higher variability in the Peruvian group than in the European one, some characteristic proteins were found by 2-DGE in some European populations, whereas it was impossible to find any in the Peruvian populations. It is concluded that the primary founders of the European populations may have an origin other than that of the Peruvian populations involved in this study.

The Globodera pallida SPRYSEC Gp-Rbp-1 gene encodes a secreted protein which induces effector-triggered immunity (ETI) mediated by the Solanum tuberosum disease resistance gene Gpa2. Nonetheless, it is not known how the Andes orogeny, the richness in Solanum species found along the Cordillera or the introduction of the nematode into Europe have affected the diversity of Gp-Rbp-1 and its recognition by Gpa2. We generated a dataset of 157 highly polymorphic Gp-Rbp-1 sequences and identified three Gp-Rbp-1 evolutionary pathways: the ‘Northern Peru’, ‘Peru clade I/European’ and ‘Chilean’ paths. These may have been shaped by passive dispersion of the nematode and by climatic variations that have influenced the nature and diversity of wild host species. We also confirmed that, by an analysis of the selection pressures acting on Gp-Rbp-1, this gene has evolved under positive/diversifying selection, but differently among the three evolutionary pathways described. Using this extended sequence dataset, we were able to detect eight sites under positive selection. Six sites appear to be of particular interest because of their predicted localization to the extended loops of the B30.2 domain and/or support by several computational methods. The P/S 187 position was previously identified for its effect on the interaction with GPA2. The functional importance of the other five amino acid polymorphisms observed was investigated using Agrobacterium transient transformation assays. None of these new residues, however, appears to be directly involved in Gpa2-mediated plant defence mechanisms. Thus, the P/S polymorphism observed at position 187 remains the sole variation sufficient to explain the recognition of Gp-Rbp-1 by Gpa2.

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