Comparing the effectiveness of real-time PCRs to simultaneously detect and identify viable Globodera pallida and G. rostochiensis.

Recently, the Commission Implementation Regulation (CIR) 2022/1192 has been published in Europe, prescribing regulations on eradication measures for quarantine potato cyst nematodes including mandated assays for species identi�cation. The CIR 2022/1192 provides a derogation on the use of Beniers et al. (2014) real-time PCR as the validation process was still ongoing. In this study, the validation of a modi�ed version of the Beniers real-time PCR was carried out, following EPPO Standard PM 7/98. Furthermore, the performance was compared to the conventional PCR of Bulman & Marshall (1997) and the Gamel et al. (2017) real-time PCR, both DNA based, which are mandated by CIR 2022/1192. Additionally, the effect of cyst viability was compared for the two real-time PCR assays. The modi�ed Beniers real-time PCR met all validation performance criteria, with analytical sensitivity of 1 living egg for both target Globodera pallida and G. rostochiensis and scoring 100% for analytical speci�city, reproducibility, robustness and diagnostic speci�city. The viability assays showed that the DNA based Gamel real-time PCR provided positive results for non-viable cysts in contrast to the modi�ed RNA based Beniers real-time PCR. Based on the results of this study, the modi�ed Beniers quali�es for the detection and identi�cation of Globodera pallida and G. rostochiensis and performs at least equally well as the other two PCR assays. For development and improvement of diagnostic protocols, it is recommended that competent authorities should be allowed to use recognized diagnostic protocols when in-house validated instead of only mandated tests as prescribed by CIR 2022/1192.


INTRODUCTION
The potato cyst nematodes (PCN), Globodera pallida and G. rostochiensis are biotrophic, sedentary endoparasitic nematodes that adversely affect potato production globally and are responsible for signi cant losses in global potato production (Sabeh et al., 2019;Gartner et al., 2021;Price et al., 2021;Orlando & Boa, 2022).The distribution is still expanding, not only in Europe but all over the world.Both species have now been reported from over 75 countries (Rowe 2022a,b;Orlando & Boa, 2023) having originated from the Andean region of South America with Europe as secondary distribution centre (Hockland et al., 2012;Price et al., 2021).PCN has the ability to survive for many years as cysts, an easily dispersed survival stage.Therefore, these nematodes have been listed on quarantine lists worldwide and are subject to strict phytosanitary regulation to prevent further spread.
In this regard, EU Council Directive 2007/33/EC, which provides regulations on control measures and prevention of PCN establishment, has been updated with the Commission Implementation Regulation (CIR) 2022/1192.Strict measures must be taken to eradicate PCN from infested potato elds in Europe.For the Netherlands, as large seed potato exporting country, responsible for about 60% of all international seed potato trade, this is very important.
The need for a quick, reliable and sensitive detection and identi cation method, even at an early stage of infestation, is of great importance to safeguard the Dutch seed potato industry.The Dutch General Inspection Service for Agricultural Seeds and Seed Potatoes (NAK), as o cial lab, is processing at least 150K soil samples a year for the detection and identi cation of PCN.These samples include an o cial detection survey of elds prior to planting of seed potatoes.For identi cation, following cyst isolation from soil, the CIR 2022/1192 stipulates the use of morphological characters combined with conventional PCR test of Bulman & Marshall (1997) and/or Skantar et al. (2007) or using the DNA based real-time PCR of Gamel et al. (2017).However, a derogation is provided by the EU to competent authorities for the use of the reverse transcription RNA based real-time PCR of Beniers et al. (2014) during a transition period ending 15 July 2024.
Numerous molecular methods to detect and identify PCN have been published (e.g.Madani et al., 2005;Toyota et al., 2008;Reid et al., 2015;Kushida & Sakai, 2022).The EPPO standard diagnostic protocol for G. rostochiensis and G. pallida PM 7/40 (5) (EPPO, 2022), recommends seven PCR based tools in combination with morphological identi cation.These include the conventional PCR of Bulman & Marshall (1997) and both the real-time PCR's of Gamel et al. (2017) and Beniers et al. (2014).The conventional PCR developed by Bulman & Marshall (1997) targets the small ribosomal subunit 18S rRNA and internal transcribed spacer (ITS1) region using a generic forward primer and two species-speci c reverse primers (van de Vossenberg et al., 2014).It should be noted that EPPO (2022) recommends using this test only in combination with morphological identi cation as the primers listed in the standard are not speci c for Globodera spp.However, van de Vossenberg et al. (2014) modi ed this conventional PCR with an optimized forward primer (ITS5_Glob) and a G. pallida speci c reverse primer (Gpalli_R) to address this issue.The DNA based Gamel et al. (2017) real-time PCR targets species-speci c microsatellites and their anking regions utilizing two separate species-speci c primer sets and probes in a multiplex Taqmanbased PCR.This real-time PCR is also able to detect target DNA isolated directly from debris material obtained after soil extraction.Beniers et al. (2014) developed a real-time PCR test for simultaneous detection of viability and species identi cation of G. rostochiensis and G. pallida, utilizing reverse transcription of the elongation factor 1-αencoding mtRNA and real-time PCR in a single reaction.Viability is determined based on the presence of the mtRNA that is rapidly broken down after death.At the same time, two species-speci c probes are used for identi cation, targeting cDNA ampli ed by a single primer set.
NAK is, as competent authority in the Netherlands, responsible for the sampling and testing of soil for the presence of PCN in elds intended for the production of seed potatoes.Annually, the NAK collects and tests approximately 150K soil samples for PCN of which around 1.4K contain PCN cysts.Already for many years, cyst vitality of PCN ndings is determined in the Netherlands, in order to monitor the effect of applied measures and control strategies.In addition, it is applied to bene t from the derogation on reduced sampling size, as provided in Annex 3, point 3c of the CIR.Therefore, NAK needs to apply a test determining both species and viability of PCN

Nematode populations
For the validation (analytical sensitivity, speci city, robustness and reproducibility) of the modi ed Beniers et al.
(2014) real-time PCR, a total of 19 populations of Globodera spp., four population of Heterodera spp.and a single population of Cactodera cacti were used (Table 1).The Globodera spp.populations included eight populations of G. rostochiensis, representing the pathotypes Ro1, Ro2,3, Ro4 and Ro5, and ten populations of G. pallida, representing the pathotypes Pa2 and Pa3, respectively.Three Heterodera populations each representing H. schachtii, H. avenae and H. trifolii were collected from various elds in the Netherlands.For the comparison of the three different PCR assays for diagnostic speci city on eld populations, 18 populations of Globodera spp., and three populations of Heterodera spp.were used (Table 5).These included pathotypes Pa2 and Pa3 of G. pallida, Ro1 and Ro2,3 pathotypes of G. rostochiensis, and 14 Globodera populations isolated from soil samples of elds with patches of poor growing potato plants which were investigated by the Dutch NPPO.These eld populations were rst identi ed by NIVIP, the Dutch national reference laboratory (NRL) based on morphology, morphometrics and the modi ed conventional PCR of Bulman & Marshall (1997) by van de Vossenberg et al. (2014).With the exception of the eld populations, all were reared in quarantine glasshouses and species identity was con rmed for all populations by morphology and morphometrics.For the viability assays, G. rostochiensis (Ro1, Ecosse (2016)) and G. pallida (Pa2, PD248 (2016)) were used.

Nucleic acid extraction
Prior to nucleic acid extractions, cysts and living second-stage juveniles (J2) and eggs (LJE) were grounded in a 1,5 ml Eppendorf tube using two 3 mm +/-1µm (G20W) dia.fat-free steel beads in 100 µl sterile water using a Retsch vibrating mill for 5 min.at 30 Hrtz.To each sample, 1 µl tomato leaf sap (30x diluted) was added to serve as internal PCR control.Total nucleic acid extractions were carried out using the Masterpure™ kit (QIAGEN, Venlo, The Netherlands) with SNAP Solution (Isogen, Life Science B.V., Utrecht, The Netherlands) magnetic beads on a KingFisher Flex-96 puri cation system (ThermoFisher Scienti c, Waltham, Massachusetts, USA) using the protocols and programme recommended by QIAGEN and ThermoFisher.Two additional controls were also included to access purity of nucleic acid extraction: (1) no added cyst to the sample to con rm purity of the kit (IC1), and (2) lysis buffer added after all samples have been loaded (IC2) to ensure no cross contamination of samples.
Modi ed real-time PCR (Beniers et al., 2014) Multiplex PCR reaction cycling conditions, primers and probes for the real-time PCR based on Beniers et al. (2014) were followed as described by EPPO (2022).For optimal implementation at the NAK, the concentrations of the components of the PCR reaction mixtures were adapted as indicated in Table 2. Additionally, primers and probes for the internal PCR control were included and the nal reaction volume was adjusted to a volume of 20 µl with PCR-grade water (Table 2).The single-step reverse-transcription real-time PCR was performed using the One Step Prime Script™ RT PCR kit (TaKaRa Bio Inc., Kusatsu, Japan) with 5 µl target RNA per reaction on an ABI Prism®7500 Sequence Detection System.Data generated was analyzed according to the manufacturer's software instructions.The PCR e ciency and nucleic acid extraction success were assessed using the mRNA of the mitochondrial nad5 gene of tomato with primers (Menzel et al., 2002) and probe as described by Botermans et al. (2013).The two additional controls (IC1 and IC2), water (no RNA; ntc) and an in-house prepared positive control (G.rostochiensis and G. pallida nucleic acid mix (Glob6)), were included in each run.Real-time PCR (Gamel et al., 2017) The Gamel et al. (2017) based multiplex real-time PCR was performed using the primers and probes as described by EPPO (2022) with slight adjustments of the reaction mixture (Table 3) for optimal performance on an ABI Prism®7500 Sequence Detection System and inclusion of the internal PCR control.The PCR was performed using the QuantiTect Multiplex PCR kit (QIAGEN, Venlo, The Netherlands) and 5 µl of target DNA per reaction using similar cycling conditions as described by EPPO (2022).The PCR e ciency and nucleic acid extraction success were assessed using the cytochrome oxidase (COX) primers and probes (Weller et al., 2000) as described by Roenhorst et al. (2005).Two additional controls (IC1 and IC2), water (no RNA; ntc) and an in-house prepared positive control (G.rostochiensis and G. pallida nucleic acid mix (Glob6)), were included in each run.(Chavornay)) and two populations of G. rostochiensis (Ro1 (Ecosse) and Ro2,3 (BC262)) were prepared and assayed.From this, the upper Ct-value limits were then calculated using the linear regression y = 36.39-2.08*ln(x)with 95% con dence limits for G. pallida, and the linear regression y = 34.12-1.78*ln(x)with 95% con dence limits for G. rostochiensis as determined by J.E. Beniers (pers. comm.),where y refers to the Cycle Threshold (Ct) value detected during the real-time PCR, and x refers to the number of viable eggs in your used sample.
The analytical speci city was determined using different populations of G. rostochiensis and G. pallida, and populations of closely related non-target nematodes (

Viability
Viability assays were performed on one population each of G. rostochiensis (Ro1 (Ecosse)) and G. pallida (Pa2 (PD248)) that were pooled together.The cysts were autoclaved for 20 min.at 121°C under 15 psi pressure.An additional subsample of cysts from both populations were kept untreated at 2-8°C.After autoclaving, cysts were divided into eight samples, four of which containing a single cyst and four containing a pool of 10 cysts,.The nonautoclaved cysts were similarly divided for each population, taking care none of the populations were mixed when the samples were prepared to con rm purity of the populations.Both autoclaved and non-autoclaved cysts samples were used for visual con rmation of cyst content (EPPO PM7/40 (5)

Validation
The results of the single egg assays using the modi ed Beniers et al. (2014) real-time PCR are indicated in Table 4.For both G. rostochiensis and G. pallida a LOD of 1 LJE was successfully obtained.Based on the linear regression formulas, the limit for considering a sample as a positive result during real-time PCR was set at a maximum of 38 cycles for G. pallida and a maximum of 34 cycles for G. rostochiensis.Table 6 shows the results per test for each sample set.For both PCR assays, the internal controls nad5 (Beniers et al., 2014) and COX (Gamel et al., 2017) were positive where applicable, indicating that nucleic acid extraction was successful and PCR conditions were correct.Additional PCR controls (IC1, IC2, Globo6 and ntc) also provided the expected results.Both PCR assays indicated that the non-treated G. rostochiensis (Ro1) and G. pallida (Pa2) populations were not cross-contaminated.For the cysts treated in the autoclave, the modi ed Beniers et al. (2014) real-time PCR provided a negative result for both target Globodera spp. in all samples assayed.In the case of the Gamel et al. (2017) real-time PCR, all samples assayed provided a positive reaction for G. pallida.Both samples Gp + rAut(B) and Gp + rAut(C), each containing a single cyst, gave a positive result for both target Globodera spp.and sample Gp + rAut(H) (10-cyst pool) was also positive for both target Globodera spp., although efforts were made to prevent cross contamination.Visual inspection of cysts showed that the non-autoclaved cysts contained numerous living eggs and second-stage juveniles for both Globodera spp., whereas the autoclaved cysts contained no to severely damaged second-stage juveniles.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 in uenced by reagents and equipment used, and that each laboratory needs to determine if a test is t for purpose under their conditions through a validation process upon implementation.).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 classi ed 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 elds affected by infestations, such as the use of resistant varieties, empty cysts frequently persist in these elds.Utilizing the testing methods outlined in the Commission proposal could lead to inaccurate positive results.
Inundation of infested elds or the use of highly resistant potato varieties on infested elds 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 elds has been used with high success to eradicate G. rostochiensis (Price et  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 signi cance, particularly when assessing the viability of PCN in treated elds.A positive result based on a DNA PCR only con rms the historic presence of the target genetic material/organism, but does not con rm the current viability and signi cance of the target organism, especially after eradication measures.Therefore, it is critical to assess the viability of cysts collected from an infested eld 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 con rmation 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 modi ed Beniers et al. (2014) real-time PCR clearly obtained negative results for non-viable cysts, as was also con rmed 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 ( eld) 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 speci city results all were conform expectations and con rmed 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 identi cation, 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 elds, even though required eradication measures have been applied, making elds 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).

DECLARATIONS
ndings.In this study, NAK aimed to further modify the RNA based real-time PCR of Beniers et al. (2014) for qualitative detection of viability and identi cation of G. rostochiensis and G. pallida in one single test, as no published molecular test is available yet.Given that no validation has yet been published for the application of the Beniers et al. (2014) real-time PCR in a routine diagnostic laboratory setup, the performance criteria of analytical sensitivity and -speci city, robustness and reproducibility of the modi ed version by NAK of this real-time-PCR was determined according to EPPO Standard PM7/98 (EPPO, 2021).Furthermore, the performance of both the modi ed Beniers et al. (2014) and Gamel et al. (2017) real-time PCR assays on eld populations were compared to the conventional PCR of Bulman & Marshall (1997) to evaluate their application in a diagnostic setup.Additionally, an assay was performed to determine the biological relevance of viability by comparing the modi ed Beniers et al. (2014) real-time PCR with the Gamel et al. (2017) real-time PCR.
compare and the diagnostic speci city of both the real-time PCR's, the modi ed Beniers et al. (2014) and Gamel et al. (2017) were compared to the conventional PCR of Bulman & Marshall (1997).Single cysts of eld populations collected from patches of poor growth, of both target Globodera spp.and non-target Heterodera spp.were analyzed as indicated above.As positive controls two known populations each of G. rostochiensis (Ro1 & Ro2,3) and G. pallida (Pa2 & Pa3) were included (Table , 2022 (appendix 10)) and for nucleic acid extraction as indicated above.To illustrate the biological importance of cyst viability both the modi ed Beniers et al. (2014) real-time PCR and Gamel et al. (2017) real-time PCR were applied for each treatment.
G. rostochiensis Ro1 population (Ecosse), Pa2 = G. pallida Pa2 population (PD248), Gp + r = mix of Ro1 and Pa2 cysts; 2 none = untreated and autoclave = autoclaving at 121°C at 15 psi; 3 Gpal = G. pallida; 4 Gros = G. rostochiensis,5 IC= internal controls without target nucleic acid; 6 Globo6 = in-house developed PCR positive control;7 ntc = PCR negative control (water); grey blocks indicate discrepancies in results of PCRs used.G. rostochiensis Ro1 population (Ecosse), Pa2 = G. pallida Pa2 population (PD248), Gp + r = mix of Ro1 and Pa2 cysts; 2 none = untreated and autoclave = autoclaving at 121°C at 15 psi; 3 Gpal = G. pallida; 4 Gros = G. rostochiensis, 5 IC= internal without target nucleic acid; 6 Globo6 = in-house developed PCR positive control; 7 ntc = PCR negative control (water); grey blocks indicate discrepancies in results of PCRs used.DISCUSSION 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) modi ed by NAK.This study included populations reared in the greenhouse and populations collected from elds where Globodera-related symptoms on potato plants were observed.As neither of the three mentioned PCR assays were tested against eld 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 -speci city, robustness and reproducibility was performed for the routine implementation of the modi ed Beniers et al. (2014) real-time-PCR.The conventional PCR of Bulman & Marshall (1997) targets the small ribosomal subunit 18S rRNA and anking 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-speci c reverse primers.The Gamel et al. (2017) real-time PCR targets speci c short DNA repeats or microsatellites and their anking regions that are unique for either G. pallida or G. rostochiensis, requiring two sets of species-speci c 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-speci c 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 modi cations and an inhouse validation were required.These modi cations mostly included changes in reagent concentrations of the PCR reaction mixtures to best t 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 su cient 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 speci city of the modi ed Beniers et al. (2014)

Table 1
Populations used for the validation of the modi ed Beniers et al. (2014) real-time PCR and the obtained results on their analytical speci city.

Table 2
Reaction mixture used for the modi ed multiplex reverse transription real-time PCR based on Beniers et al. (2014).

Table 3
Adapted reaction mixture used for the multiplex real-time PCR based on Gamel et al. (2017).
Validation modi edBeniers et al. (2014) real-time PCR The analytical sensitivity is a fundamental parameter to de ne the limit of detection (LOD) needed to reliably detect target DNA/RNA in a sample.To evaluate the LOD of the modi ed Beniers et al. (2014) real-time PCR, triplicate samples consisting of single viable eggs of two populations of G. pallida (Pa2 (PD248) and Pa3

Table 1 )
Beniers et al. (2014)bustness and reproducibility, the modi edBeniers et al. (2014)real-time PCR was performed in a blind test on eight different occasions using ve different ABI Prism®7500 Sequence Detection Systems by two independent technicians.For this purpose, the populations were divided over 19 samples, with known numbers of LJE and anonymized.Samples were randomly divided between the independent technicians with at least one sample assayed in triplicate.A Positive Isolation Control (PIC), containing a mixture of 12 LJE of both target Globodera spp. was included for each repeat of the assays.

Table 4
Beniers et al. (2014)alues obtained by the modi ed NAKBeniers et al. (2014)real-time PCR to de ne the limit of detection (LOD) with single eggs of each target species.The analytical speci city of the modi edBeniers et al. (2014)was tested on both target Globodera spp., and on six non-target species.The target Globodera spp.consisted of six populations for G. rostochiensis and eight populations of G. pallida.The non-target species included one population each of Cactodera cacti, G. tabacum, Heterodera betae, H. glycines, H. schachtii and H. trifolii.The modi edBeniers et al. (2014)test scored 100% for analytical speci city (Table1).None of the non-target species gave a false positive result and none of the target Globodera spp.gave a false negative result.Results obtained for the diagnostic speci city are presented in Table5.For both real-time PCR's, the internal controls nad5 for the modi edBeniers et al. (2014)and COX for Gamel et al. (2017) real-time PCR's were positive where applicable.For all three PCR assays used, both the PCR positive and negative controls (IC1, IC2, Globo6 and ntc) provided the expected results.Both populations of G. rostochiensis (Ro1 and Ro2,3) and of G. pallida (Pa2 and Pa3) were correctly identi ed by all three PCR assays.The three Heterodera eld populations (H.avenae, H. schachtii and H. trifolii) all tested negative in all three PCR assays.Comparison of the results of the single cyst samples obtained from the 14 soil samples of the NPPO-NL gave no discrepancies between the three PCR assays used.All three PCR assays indicate the presence of G. pallida in 8 samples and the presence of G. rostochiensis in 6 samples.All results were in accordance with the morphological and molecular identi cations of NIVIP.
1 number of living eggs; 2 Gros = G. rostochiensis; 3 Gpal = G. pallida; 4 IC = internal controls without target nucleic acid; 5 Globo6 = in-house developed PCR positive control; 6 ntc = PCR negative control (water).To ensure the reproducibility and robustness of the modi ed Beniers et al. (2014) real-time PCR, the assays were run on ve different ABI Prism®7500 Sequence Detection Systems by two independent technicians.All assays provided a positive result for the target Globodera spp. with Ct values ranging between 17,9 to 33,2 for G. rostochiensis and between 19,2 to 33,4 for G. pallida with no false negative or false positive results.Diagnostic city

Table 5
Comparison of the PCR results obtained from eld populations and soil samples taken from patches with poor potato plant growth to determine the diagnostic speci city.

Table 6
Results of the two real-time PCR's performed on cysts subjected to treatment for viability determination.
Bulman & Marshall (1Gartner et al., 2021;Orlando & Boa, 2023el et al. (2017)PCRs were compared in this study to the conventional PCR ofBulman & Marshall (1997)and the identi cation results obtained by NIVIP.All tests were properly implemented and executed, internal controls, and positive and negative controls scored as expected.For the eld populations, all PCR assays consistently yielded the same positive identi cations for both G. pallida and G. rostochiensis.Diagnostic speci city 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 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 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 identi cation of PCN in infested elds is of utmost