In this study, the performance of the two PCR assays mandated by new European legislation (CIR 2022/1192), based on Bulman & Marshall (1997) and Gamel et al. (2017) were compared with the RNA real-time PCR assay based on Beniers et al. (2014) modified by NAK. This study included populations reared in the greenhouse and populations collected from fields where Globodera-related symptoms on potato plants were observed. As neither of the three mentioned PCR assays were tested against field populations during their development, it is important to determine their performance in a routine laboratory setup. At the same time, a validation based on analytical sensitivity and -specificity, robustness and reproducibility was performed for the routine implementation of the modified Beniers et al. (2014) real-time-PCR.
The conventional PCR of Bulman & Marshall (1997) targets the small ribosomal subunit 18S rRNA and flanking internal transcribed spacer (ITS1) region, which was further improved by van de Vossenberg et al. (2014). This PCR distinguishes between G. pallida and G. rostochiensis based on DNA fragment sizes observed after gel electrophoresis caused by differences in the binding sites for the species-specific reverse primers. The Gamel et al. (2017) real-time PCR targets specific short DNA repeats or microsatellites and their flanking regions that are unique for either G. pallida or G. rostochiensis, requiring two sets of species-specific primers and probes. In contrast, the reverse transcription real-time PCR developed by Beniers et al. (2014) targets the mtRNA of the elongation factor 1-α gene region, by using a single primer pair and two species-specific probes to target the cDNA. What distinguishes this mtRNA based PCR assay from the other two DNA based PCR assays, is its ability to indicate the viability of the cysts collected. In principle, a positive result can only be obtained by the presence of the target mtRNA, indicating viable eggs or J2 juveniles in the sample tested.
For a successful implementation of the Beniers et al. (2014) real-time PCR at NAK, some modifications and an in-house validation were required. These modifications mostly included changes in reagent concentrations of the PCR reaction mixtures to best fit the capacity at the NAK. Validation of these changes, according to EPPO Standard PM7/98 (EPPO, 2021) were carried out successfully at the NAK. Analytical sensitivity showed that only a single egg is sufficient to obtain a positive result, an improvement on the limit of detection indicated by Beniers et al. (2014) of 30 JLE for both target Globodera spp. The analytical specificity of the modified Beniers et al. (2014) was also successfully proven, with no false positive result obtained for the non-target populations included. The reproducibility and robustness were also completed successfully with expected results obtained for all samples tested. However, given the wide range of Ct values for the same samples, the authors recognize that performance criteria of molecular tests may be influenced by reagents and equipment used, and that each laboratory needs to determine if a test is fit for purpose under their conditions through a validation process upon implementation.
Diagnostic specificity of both the Beniers et al. (2014) and Gamel et al. (2017) PCRs were compared in this study to the conventional PCR of Bulman & Marshall (1997) and the identification results obtained by NIVIP. All tests were properly implemented and executed, internal controls, and positive and negative controls scored as expected. For the field populations, all PCR assays consistently yielded the same positive identifications for both G. pallida and G. rostochiensis. Diagnostic specificity ensures that the results obtained are accurate and reliable, regardless of the used test, reducing the chances of misdiagnosis. The results achieved by NAK show that all three different PCR tests can be implemented with high reliability in a diagnostic lab.
Viability of the cysts is important in order to assess whether eradication measures, as now mandated by CIR 2022/1192, applied by seed potato growers have been effective given that PCN cysts can persist in soil for some time (Price et al., 2020; Gartner et al., 2021; Orlando & Boa, 2023). However, the current PCR assays included in CIR 2022/1192 do not provide the capability to differentiate between living and dead cysts in testing. Dead cysts pose no risk of disease or spread, and can provide a positive indication of successful eradication measures. However, in a test lacking the ability to discriminate between living and dead cysts, the dead cysts can be incorrectly classified as risk-posing cysts. The proposal is to incorporate into the regulation a testing method that can differentiate between living and dead cysts. It is imperative to include detection assays which can distinguish viability of cysts. After implementing control measures in fields affected by infestations, such as the use of resistant varieties, empty cysts frequently persist in these fields. Utilizing the testing methods outlined in the Commission proposal could lead to inaccurate positive results.
Inundation of infested fields or the use of highly resistant potato varieties on infested fields for ware or starch potatoes are mandatory measures. Inundation has proven an effective eradication strategy, with loss of viability of PCN cyst content (Runia et al., 2014). Nevertheless, non-viable cysts were still isolated after inundation (Runia et al., 2014; Berger et al., 2023). The use of resistant potato varieties in infested fields has been used with high success to eradicate G. rostochiensis (Price et al., 2020; de Boer et al., 2021; Gartner et al., 2021; Mwangi et al., 2021; Orlando & Boa, 2023). However, some G. pallida populations can overcome host resistance, resulting in more virulent populations (Fournet et al., 2016; Eoche-Bosy et al., 2017; Beniers et al., 2018; Mwangi et al., 2019; Price et al., 2020; Gartner et al., 2021). Therefore, accurate and fast identification of PCN in infested fields is of utmost importance. An aspect that should be taken into consideration is the viability of PCN cysts, especially when applying inundation as eradication measure, as non-viable cysts will remain in the soil. The possibility exists that the mandatory DNA based PCR assays as prescribed by CIR 2022/1192, give a positive indication of PCN, even though none of the cysts are viable and therefore pose no risk, questioning the biological relevance of a positive result obtained by DNA based PCR assays as diagnostic tool.
A positive result based on a RNA based PCR holds substantial biological significance, particularly when assessing the viability of PCN in treated fields. A positive result based on a DNA PCR only confirms the historic presence of the target genetic material/organism, but does not confirm the current viability and significance of the target organism, especially after eradication measures. Therefore, it is critical to assess the viability of cysts collected from an infested field if the use of the DNA based PCR’s of Bulman & Marshall (1997) and/or Gamel et al., (2017) are mandatory following CIR 2022/1192. This might require an additional step of prior visual confirmation of viable cyst content and the direct testing of soil debris is therefore not recommended.
The viability tests done in this study clearly showed that the DNA within none-viable PCN cysts, after autoclaving, could still serve as a positive target for the DNA based real-time PCR of Gamel et al. (2017). Therefore, the direct use of Gamel et al. (2017) real-time PCR (or other DNA based molecular tests), as recommended by the CIR 2022/1192, on soil debris after cyst isolation should be applied with care. It is recommended, also by EPPO (EPPO, 2022), that the cysts are evaluated for living content prior to performing a molecular test. In comparison, the modified Beniers et al. (2014) real-time PCR clearly obtained negative results for non-viable cysts, as was also confirmed by visual inspection of the cyst content. It is, however, worrisome that the Gamel et al. (2017) real-time PCR provided a positive result for both target Globodera spp., where a single non-viable cyst was used, Further research with (field) populations and viability testing at the NAK is needed to investigated the cause of this double positive signal as the positive controls within the viability test and diagnostic specificity results all were conform expectations and confirmed the validation data in Gamel et al. (2017).
Given that the EPPO standard protocol for G. rostochiensis and G. pallida PM 7/40 (5) (EPPO, 2022), recommends seven validated PCR based tools which can be used for detection and identification, it is remarkable that the CIR 2022/1192 limits the competent authorities to only two of these PCR assays. With the rapid advances in molecular technologies, this limitation could severely impede new developments for more effective tools. Furthermore, the persistence of non-viable PCN cysts could provide a misleading indication of the presence of PCN in previously infested fields, even though required eradication measures have been applied, making fields unnecessarily unavailable for potato cultivation. Therefore, it is recommended that competent authorities should be provided with the freedom to use relevant internationally recognized diagnostic protocols when in-house validated according to EPPO standards and accredited by the EU-bodies (EPPO, 2021).