Eur J Plant Pathol (2018) 151:757–766 https://doi.org/10.1007/s10658-017-1411-6 Euphresco Sendo: An international laboratory comparison study of molecular tests for Synchytrium endobioticum detection and identification Bart van de Vossenberg & Marcel Westenberg & Ian Adams & Olga Afanasenko & Ani Besheva & Margriet Boerma & James Choiseul & Toos Dekker & Kerstin Flath & Marga van Gent-Pelzer & Kurt Heungens & Anatolii Karelov & Ilona Kibildiene & Jaroslaw Przetakiewicz & Alexandra Schlenzig & Vera Yakovleva & Gerard van Leeuwen Accepted: 25 December 2017 /Published online: 7 February 2018 The Author(s) 2018. This article is an open access publication Abstract An international test performance study (TPS) 2005), and van Gent-Pelzer et al. (European Journal of was organised to generate validation data for three mo- Plant Pathology, 126, 129-133, 2010) for the detection lecular Synchytrium endobioticum tests: van den Boogert of S. endobioticum, and the pathotype 1(D1) identifica- et al. (European Journal of Plant Pathology 113, 47–57, tion test described by Bonants et al. (European Journal Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10658-017-1411-6) contains supplementary material, which is available to authorized users. : : B. van de Vossenberg (*) M. Westenberg M. Boerma G. van Leeuwen Hilbrands Laboratorium BV, Kampsweg 27, 9418 PD Wijster, Dutch National Plant Protection Organization, National The Netherlands Reference Centre, Geertjesweg 15, 6706EA, Wageningen, e-mail: email@example.com The Netherlands e-mail: firstname.lastname@example.org J. Choiseul Department of Agriculture, Food and the Marine, Backweston M. Westenberg Campus, Celbridge, Co. Kildare, Ireland e-mail: email@example.com e-mail: firstname.lastname@example.org G. van Leeuwen T. Dekker e-mail: email@example.com NAK, Randweg 14, 8304 ASEmmeloord, the Netherlands e-mail: firstname.lastname@example.org K. Flath I. Adams Julius Kühn-Institut, Stahnsdorfer Damm 81, Fera Science Ltd., Sand Hutton, York YO41 1LZ, UK 14532 Kleinmachnow, Germany e-mail: email@example.com e-mail: firstname.lastname@example.org O. Afanasenko M. van Gent-Pelzer All Russian Research Institute for Plant Protection, Podbelsky sh. Wageningen Plant Research, BU Biointeractions & Plant Health, 3, Pushkin, Saint Petersburg, Russia Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands e-mail: email@example.com e-mail: firstname.lastname@example.org A. Besheva K. Heungens Central laboratory for plant quarantine, 120, N. Moushanov Blvd, Institute for Agricultural and Fisheries Research, Plant Unit, Burg. 1330 Sofia, Bulgaria Van Gansberghelaan 96, 9820 Merelbeke, Belgium e-mail: email@example.com e-mail: firstname.lastname@example.org 758 Eur J Plant Pathol (2018) 151:757–766 of Plant Pathology, 143, 495-506, 2015). Two TPS (galls or warts) in host tissues of susceptible potato rounds were organised focussing on different test matri- cultivars resulting in yield losses up to 100% ces, i.e. round 1: warted potato tissue, and round 2: (Hampson 1993). Robust resting spores are formed in resting spore suspensions. When using the tests for de- the warted tissue and are released into the surrounding tection and identification of S. endobioticum in warted soil when host tissue decays. These resting spores, potato tissue, no significant differences were observed which can remain viable and infectious in soil for de- for diagnostic sensitivity, diagnostic specificity, overall cades (Laidlaw 1985; Przetakiewicz 2015a), together accuracy, analytical sensitivity and robustness. When with the lack of successful chemical control agents using the tests for detection and identification of (Hampson 1993), present a major challenge to potato S. endobioticum in resting spore suspensions, the van production. den Boogert and van Gent-Pelzer tests significantly out- To date, 39 pathotypes of the fungus have been perform the Bonants test for diagnostic sensitivity and described (Baayen et al. 2006, Przetakiewicz 2015b) diagnostic specificity. For overall accuracy and analyti- and phytosanitary measures heavily rely on pathotype cal sensitivity, the van Gent-Pelzer significantly outper- identification. The main focus of the current version of forms the van den Boogert and Bonants tests and is the European and Mediterranean Plant Protection Orga- regarded as the test of choice when identifying nization (EPPO) S. endobioticum standard PM7/28 S. endobioticum from resting spores. Tests regarded fit (EPPO 2004) lies with pathotype identification using for purpose for routine testing of wart material and different bioassays, and no molecular tests for pathogen resting spore suspensions are proposed for the update detection or identification are included. We aim to fill of EPPO standard PM7/28(1) Synchytrium this gap by generating validation data for three molecu- endobioticum. lar tests, including both DNA extraction and amplifica- tion, in an international test performance study (TPS). Keywords Validation Test Performance Study Two TPS rounds were organised focussing on differ- ent test matrices (i.e. warted potato tissue in round 1, and Inter-laboratory comparison study EPPO Diagnostic Standard Potato wart disease resting spore suspensions in round 2). TPS results and additionally generated data were used to evaluate the following performance criteria: analytical sensitivity, Introduction analytical specificity, diagnostic sensitivity, diagnostic specificity, repeatability and robustness following EPPO The cosmopolitan soil-borne obligate biotrophic fungus standard PM7/98 (EPPO 2014a). A draft version of the Synchytrium endobioticum (Schilb.) Perc. is the causal EPPO standard on the organisation of interlaboratory agent of potato wart disease and is considered one of the comparison studies (EPPO 2014b) was used for guid- ance during the TPS setup. most important quarantine organisms of cultivated po- tatoes (Smith et al. 1997, Obidiegwu et al. 2014). Upon Three molecular tests for S. endobioticum detec- tion were described in literature when setting up the infection, S. endobioticum induces tumor-like growth A. Karelov A. Schlenzig Institute of Plant Protection, 33 Vasylkivska Str, Kiev 3022, Science and Advice for Scottish Agriculture, 1 Roddinglaw Road, Ukraine Edinburgh EH12 9FJ, UK e-mail: email@example.com e-mail: firstname.lastname@example.org I. Kibildiene V. Yakovleva The State Plant Service, Phytosanitary Research Laboratory All-Russian Plant Quarantine Center, Pogranichnaya 32, Bykovo, (Division), Ministry of Agriculture, Sukileliu str. 9A, LT - 140150 Ramenskoe region, Moscow, Oblast, Russia 11352 Vilnius, Lithuania e-mail: Yakovleva_va@mail.ru e-mail: email@example.com J. Przetakiewicz Plant Breeding and Acclimatization Institute, National Research Institute, Radzikow, 05-870 Blonie, Poland e-mail: firstname.lastname@example.org Eur J Plant Pathol (2018) 151:757–766 759 TPS; a conventional PCR amplifying 543 bp of the 2(G1), 6(O1), 18(T1), and 38(Nevsehir) infected pota- internal transcribed spacer (ITS) region (Niepold toes. Cuttings of warted tissue from a single isolate was and Stachewicz 2004); a conventional PCR ampli- used to prepare several TPS samples. For instance 2(G1) fying 472 bp of the ITS region (van den Boogert warts (MB08) were used to prepare sample 1, sample 9 et al. 2005); and a real-time PCR TaqMan test am- and the return sample in TPS round 1 (Table 1). Healthy plifying 84 bp of the internal transcribed spacer 2 potatoes of cv. BEersteling^ and warts were cut in (ITS2) (van Gent-Pelzer et al. 2010). A short informal portions of approximately 100 mg, added to 2 mL questionnaire held among laboratories working with lyophilisation ampoules (VWR, Radnor, USA) and S. endobioticum indicated that tests described by van frozen 16 h at −80 °C prior to lyophilisation with a den Boogert et al.andvan Gent-Pelzer et al.were most BenchTop 4 K BTXL-75 freeze-dryer (VirTis, frequently used. For that reason both tests were included Warminster, USA). Ampoules were closed under in this study together with a recently developed real- vacuum and topped off with a tear-away crimp cap time PCR test for S. endobioticum pathotype 1(D1) (VWR, Radnor, USA). identification (Bonants et al. 2015), which was unpub- In the second TPS round, resting spore suspensions lished at the time of TPS preparation. The latter test, were provided. The unknown sample set consisted of targeting a pathotype 1(D1) associated single nucleotide molecular grade water (MGW; Sigma, Saint Louis, polymorphism (SNP), is the first example of molecular USA) used for resting spore suspension preparation, 5 −1 S. endobioticum pathotype identification using real-time and undiluted (approximately 5.0 × 10 spores mL ) PCR and consists of a duplex test targeting pathotype and two 10-fold dilutions of S. endobioticum pathotype 1(D1) and non-1(D1) pathotypes. In addition to the 1(D1) and 6(O1) resting spore suspensions (Table 1). specific tests, a TaqMan test targeting the plant COI Resting spores were isolated from fresh warts with gene was used as an internal control (Mumford et al. 75 μm and 45 μm mesh sieves. A heat treatment 2004). In this paper, tests described by van den Boogert (15 min at 95 °C) was performed on the resting spore et al., van Gent-Pelzer et al.,Bonants et al. and Mum- suspension stocks to render the resting spores non-via- ford et al. are referred to as Sendo PCR, Sendo TaqMan, ble. Preliminary tests performed on heat treated and 1(D1) and non-1(D1) TaqMan, and COX TaqMan non-heat treated resting spore suspensions showed that respectively. the heat treatment had no effect on PCR success (data Tests regarded as fit for purpose based on the perfor- not shown). For each sample, 10 μL heat treated resting mance criteria determined in this study were suggested spore suspensions or MGW was added to a 1.5 mL for addition to the update of PM7/28. screw cap tube (VWR, Radnor, USA). Sample set homogeneity was determined by analysing 10 aliquots per sample using all tests. Materials and Methods TPS organisation Participants Participants were provided with most items needed for Fifteen laboratories involved in research or diagnostic TPS participation to minimise factors that could influ- activities for S. endobioticum with at least two years of ence test performance. In both TPS rounds, participants experience with molecular techniques participated in the were provided with positive and negative amplification study. A workshop with training session was organised controls (PAC and NAC), positive and negative isola- before the start of the first TPS round to familiarise tion controls (PIC and NIC), and 10 unknown samples. participants with the TPS setup and the tests included. In addition, TPS packages contained aliquots of the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany), Sample set preparation primers and probes, a return sample, 15 mL MGW for reaction mix preparation, transport documents for the Warted potato tissue was used as starting material for return sample (aliquot of sample wart 2(G1), round 1 DNA extraction in the first TPS round. The sample set only) and an instruction booklet. Test descriptions were consisted of randomised healthy potato pieces and wart provided following the format for EPPO diagnostic pieces taken from S. endobioticum pathotypes 1(D1), protocols to not only determine the test performance, 760 Eur J Plant Pathol (2018) 151:757–766 Table 1 TPS samples from round 1 (wart material) and round 2 (resting spores), and their assigned qualitative values based on homogeneity test results TPS round Control/sample Material pathotype/ cultivar Strain Assigned evalues Sendo PCR Sendo TaqMan 1(D1) TaqMan 1& 2 NAC MGW –– – – – 1 & 2 PAC1 1(D1) Wart DNA 1(D1) MB42 + + 1(D1) −2 1 & 2 PAC2 1(D1) 10 PAC1 1(D1) 1(D1) MB42 + + 1(D1) 1 & 2 PAC1 non-1(D1) Wart DNA 2(G1) MB08 + + Non-1(D1) −2 1 & 2 PAC2 non-1(D1) 10 PAC1 non-1(D1) 2(G1) MB08 + + Non-1(D1) 1 Sample 1 wart tissue 2(G1) MB08 + + Non-1(D1) Sample 2 wart tissue 1(D1) MB42 + + 1(D1) Sample 3 wart tissue 18(T1) MB86 + + Non-1(D1) Sample 4 wart tissue 38(Nevsehir) MB56 + + Non-1(D1) Sample 5 healthy potato Eersteling –– – – Sample 6 wart tissue 6(O1) MB10 + + Non-1(D1) Sample 7 wart tissue 18(T1) MB86 + + Non-1(D1) Sample 8 wart tissue 6(O1) MB10 + + Non-1(D1) Sample 9 wart tissue 2(G1) MB08 + + Non-1(D1) Sample 10 wart tissue 38(Nevsehir) MB56 + + Non-1(D1) Sample 11 wart tissue 1(D1) MB42 + + 1(D1) Sample 12 healthy potato Eersteling –– – – NIC healthy potato Eersteling –– – – PIC wart tissue 1(D1) MB42 + + 1(D1) Return Sample Wart tissue 2(G1) MB08 + + Non-1(D1) b c 2Sample1 5sps 1(D1) MB42 Und Und Und Sample 2 5000 sps 6(O1) MB10 + + Non-1(D1) Sample 3 MGW –– – – – Sample 4 50 sps 6(O1) MB10 Und Und Und Sample 5 5 sps 6(O1) MB10 Und Und Und Sample 6 500 sps 1(D1) MB42 + + 1(D1) Sample 7 MGW –– – – – Sample 8 500 sps 6(O1) MB10 + + Non-1(D1) Sample 9 50 sps 1(D1) MB42 Und Und Und Sample 10 5000 sps 1(D1) MB42 + + 1(D1) NIC MGW –– – – – PIC 5000 sps 1(D1) MB42 + + 1(D1) a. molecular grade water, b. resting spores per sample (10 μL molecular grade water), c. Undetermined: samples below the limit of detection, and with repeatability scores <100% but also the user-friendliness of the test description homogeneity tests were taken from the same batch as proposed for the update of EPPO PM 7/28(1). The test provided to the participants. TPS samples were description provided to the TPS participants is presented regarded as suitable when resulting in the expected in the supplementary material (SI 1). qualitative results and producing Ct values with Prior to shipment of TPS packages, aliquoted sam- standard deviations <3.3. ples, primers, probes and extraction kits were tested for Upon receipt of the TPS package, partners had to homogeneity. Reagents and extraction kits used for the send the return sample to the TPS organisers who Eur J Plant Pathol (2018) 151:757–766 761 extracted DNA from the samples and analysed them pathogen cannot be quantified from wart material, a using the Sendo TaqMan. Ct values obtained from the relative infection rate was used to express the limit of return samples were used to determine if sample ship- detection (LOD). Naturally infected potato wart pieces ment influenced the TPS results. were regarded to have a relative infection rate of 100%. When TPS partners obtained unclear or contradictory For resting spore suspensions, the amount of resting results for the included controls, a spare sample set had spores per sample is used to express the LOD. Wart to be used to repeat the tests. For each sample analysed, material of fifteen S. endobioticum strains covering five participants had to provide qualitative test results, gel- different pathotypes were analysed to determine the images for the conventional Sendo PCR and Ct values analytical specificity (i.e. performance of a test with for the TaqMan tests. In addition participants had to state regard to cross-reactions with non-targets; EPPO 2017) if the protocols were strictly followed, and which grind- of the different tests. Materials used to determine the ing procedure and thermocyclers were used. analytical sensitivity and analytical specificity were test- ed as described in the supplementary information (SI 1). Performance criteria Using the data generated by TPS participants, diagnostic Results sensitivity, diagnostic specificity, accuracy, repeatability and robustness were determined for each test-matrix Homogeneity and stability results combination. Data from partners that failed to produce correct results for the provided controls were excluded Potato wart sample sets used in TPS round 1 produced from the analysis. Positive agreement (PA), negative homogenous test results for all samples in all tests (SI table 1). Mean Ct values ranged from 15.5 to 19.6 with agreement (NA), positive deviation (PD) and negative deviation (ND) (EPPO 2014a, b) of results provided by standard deviations ranging from 1.0 to 2.2. Resting TPS participants relative to the assigned values based on spore suspensions (round 2) produced homogenous test the homogeneity tests were calculated. Diagnostic sen- results with the Sendo TaqMan for the undiluted and sitivity (PA/(PA + ND), diagnostic specificity (NA/ 10× diluted samples for both pathotypes, and with the (NA + PD) and accuracy ((PA + NA)/(PA + NA + PD + 1(D1) TaqMan for undiluted samples of pathotype ND)) are expressed in percentages and provide insight 6(O1). Other sample-test combinations failed to produce in false negative results, false positive results and the the expected qualitative results for all aliquots tested. In overall performance of a test respectively. Each TPS the analysis of the TPS results, samples with 5000 and participant received multiple aliquots of the same orig- 500 resting spores per sample were regarded positive for inal sample. Qualitative results of these biological all tests and were used to determine the performance criteria of the tests. duplicates and triplicates were used to calculate the repeatability per TPS partner per test-matrix combi- Return samples analysed with the Sendo TaqMan nation. Robustness was determined by inventorying were used to determine the sample stability. Analysis variations to the protocols (i.e. disruption methods of the return samples produced Ct values similar to the and thermocyclers used) to determine if they influ- Ct values obtained for the homogeneity tests indicating enced test accuracy. For disruption methods, quali- that sample shipment did not negatively influence the tative and quantitative results of the Sendo TaqMan TPS results (Ct : 20.7, Ct mean homogeneity tests mean return were used, whereas for thermocyclers qualitative :19.6, p Students T-test: 0.284). samples results of the positive amplification controls were used to determine the robustness. Sendo TaqMan: Ct cut-off value For wart material the analytical sensitivity was deter- mined with dilutions of DNA from infected warts in Analysis of TPS round 1 data showed that late Ct values DNA from healthy potato using seven samples covering were obtained in the Sendo TaqMan in 20 of the 44 five pathotypes. For resting spore suspensions the ana- healthy potato samples (Fig 1). A Ct cut-off value was lytical sensitivity was determined with dilutions of rest- determined to distinguish false positive results from ing spore suspensions using five samples covering two truly positive samples for this test-matrix combination. pathotypes. As the presence of the non-culturable Qualitative data provided by TPS participants and 762 Eur J Plant Pathol (2018) 151:757–766 Fig. 1 TPSround1(wart material) Ct values obtained by TPS participants (♦)for the Sendo TaqMan, and the 1(D1) TaqMan (pathotype 1(D1) test, and non- pathotype 1(D1) test). Horizontal blue lines represent the mean Ct values obtained from the homo- geneity tests. Corresponding ±1 SD, ±2 SD and ±3 SD values are represented by green, orange and red horizontal lines, respectively. Negative samples are assigned the value B0^ homogeneity results were used to calculate the mean from 95.4% to 97.2% are obtained (Table 2). For diag- false-positive Ct value and corresponding standard de- nostic specificity (the percentage of samples with ab- viation. Three standard deviations were subtracted from sence of the target that test negative) the Sendo PCR, the mean false positive Ct value resulting, after rounding down to the nearest natural number, in a Ct cut-off value Table 2 Diagnostic sensitivity, diagnostic specificity and overall of 30. Performance criteria presented in this paper are accuracy values obtained using TPS results based on the cut-off value of 30 unless stated otherwise. a b c TPS round Test DSens DSpec Acc A Ct cut-off value is not needed when testing resting spores suspensions. 1 (wart material) Sendo PCR 96.3% 100% 97.2% Sendo TaqMan 97.2% 100% 97.9% Diagnostic sensitivity, diagnostic specificity 1(D1) TaqMan 95.4% 100% 96.5% and accuracy 2 (resting spores) Sendo PCR 68.0% 100% 73.9% Sendo TaqMan 76.7% 100% 85.4% Using wart material for S. endobioticum detection, di- 1(D1) TaqMan 45.7% 77.1% 61.4% agnostic sensitivity (the percentage of sample with pres- a. diagnostic sensitivity, b. diagnostic specificity, c. accuracy ence of the target that test positive) values ranging Eur J Plant Pathol (2018) 151:757–766 763 Sendo TaqMan and 1(D1) TaqMan yield 100% correct wart 1(D1), and healthy potato) were used to determine results. No significant differences (2-sample binomial the qualitative and quantitative influence of the disrup- tests) for diagnostic sensitivity (p ≥ 0.344), diagnostic tion method applied. No significant differences are specificity (p = 1.0) and accuracy (p ≥ 0.461) are found found based on qualitative test results. Manual disrup- between the different tests when testing wart material. tion of the samples resulted in higher Ct values for the All wart samples analysed resulted in correct results for Sendo TaqMan (+3.1) and 1(D1) TaqMan (+3.4). Data the internal COX control with an average Ct value of obtained in the second TPS round could not be used to 19.3 and standard deviation of 1.9. determine the robustness when using the tests with When testing resting spores, the highest values for resting spores as starting material as only mechanical diagnostic sensitivity, diagnostic specificity and overall disruption methods were used (SI Table 3). accuracy are obtained with the Sendo TaqMan (Table 2). Expected qualitative test results for the conventional The Sendo PCR and Sendo TaqMan significantly out- PCR test were obtained using the following perform the 1(D1) TaqMan for diagnostic sensitivity (2- thermocyclers: Peltier PTC-200 (MJ research), sample binomial test, p ≤ 0.05) and diagnostic specific- GeneAmp PCR System 9700 (Applied Biosystems), ity (p = 0.003). The Sendo TaqMan significantly outper- GeneAmp PCR System 2720 (Applied Biosystems), forms both the Sendo PCR and 1(D1) TaqMan for Mastercycler personal (Eppendorf), C1000 (Bio-Rad), overall accuracy (p <0.030). Veriti 96-well thermal cycler (Applied Biosystems). Ex- pected qualitative test results for the real-time PCR tests Repeatability were obtained using the following real-time PCR sys- tems: 7300 Real-Time PCR System (Applied Biological duplicates and triplicates tested by TPS part- Biosystems), 7900HT Fast real-time PCR system (Ap- plied Biosystems), ABI 7500 Real-time PCR system ners were used to calculate the repeatability of the different tests per participant for each test matrix (SI (Applied Biosystems), CFX96 (Bio-Rad), Mastercycler Table 2). For TPS round 1, results of 13 partners were ep realplex (Eppendorf), and Stratagene Mx3005P included in the analysis, and a total of 52 repeatability (Agilent genomics). samples were analysed. Similar repeatability results are obtained for all three tests. With an average repeatability Analytical sensitivity of 98%, the TPS participants produced repeatable results when analysing wart material. For the second TPS The lowest amount of the target at which all samples round, 14 to 28 repeatability samples tested for the produce a positive results is regarded as the LOD which Sendo PCR (14 partners), Sendo TaqMan (12 partners), were determined for both test matrices at intra- and 1(D1) TaqMan (seven partners) were analysed. laboratory level (SI Table 4). When using wart material When testing resting spore suspensions with 5000 as input, the Sendo PCR, Sendo TaqMan, Bonants test spores per sample, the Sendo TaqMan and Sendo PCR for pathotype 1(D1) samples, and Bonants test for non- significantly outperform the 1(D1) TaqMan (2-sample pathotype 1(D1) samples have a LOD at a relative binomial test, p ≤ 0.029) with an average 83% repeat- infection rate of 1% (i.e. a 100× dilution of a naturally ability. Even though the Sendo PCR statistically outper- infected wart). Using resting spore suspensions, only the forms the 1(D1) TaqMan, the average reproducibility of Sendo TaqMan, and the non-1(D1) TaqMan produced both test is poor when analysing resting spore suspen- consistent results for all subsamples at 500 and 5000 sions (64% and 29% respectively). resting spores per 10 μL sample respectively. The Sendo PCR, and the 1(D1) TaqMan were not sensitive enough Robustness to detect the pathogen in all subsamples with 5000 resting spores per 10 μL sample. The LOD for the latter In TPS round 1, eleven participants specified the dis- two tests lies higher than 5000 spores per 10 μLsample. ruption method used (i.e. manual versus mechanical disruption). Two partners manually grinded samples Analytical specificity preceding the DNA extraction, whereas nine partners indicated they used a mechanical disruption method. Analytical specificity is the ability of a test to detect a Samples tested as biological duplicate or triplicate (i.e. particular target, rather than others. In case of the Sendo 764 Eur J Plant Pathol (2018) 151:757–766 PCR and Sendo TaqMan, S. endobioticum (covering all communication Kurt Heungens, ILVO, Belgium). The pathotypes) is the target, whereas in the 1(D1) TaqMan Sendo TaqMan produced late Ct values for some truly only S. endobioticum pathotype 1(D1) strains are the negative samples, which could not be reproduced with target. In the first two tests, healthy potato material the Smith test. With the Smith test producing slightly serves as non-target material, whereas in the third test lower Ct values in general, this suggests late Ct values both healthy potato material and S. endobioticum strains are the result of non-specific annealing of primers and of pathotypes other than 1(D1) are non-targets. Wart probes in the Sendo TaqMan rather than as a result of material of 15 strains was analysed to determine their contamination. We propose to include the cut-off value reaction in the different tests (SI Table 5). For the Sendo in the EPPO standard. No false negative results were PCR and Sendo TaqMan all strains produced results as obtained when using the cut-off value. Laboratories expected; i.e. positive in case S. endobioticum was implementing the Sendo TaqMan have to determine present in the sample. For the 1(D1) TaqMan however, the need of a Ct cut-off value for their diagnostic one Swedish pathotype 1(D1) isolate produced a result workflow through the process of verification. consistent with non-pathotype 1(D1) samples; i.e. a When using the tests for detection and identification false negative result. Other isolates produced results as of S. endobioticum in warted potato tissue (TPS round expected in the 1(D1) TaqMan. 1), no significant differences were observed for diag- nostic sensitivity, diagnostic specificity and overall ac- curacy and the tests are regarded equal. Also, the tests Discussion and Conclusions show an equal performance in terms of analytical sen- sitivity using this test matrix. All tests were found to be Three tests were selected for the detection and identifi- robust for the disruption method used. The second TPS round proved to be more challeng- cation of S. endobioticum, the causal agent of potato wart disease. Tests were validated in an international ing than the first since the sample set provided contained TPS with fifteen participants for two test matrices: wart- resting spore suspensions close or below the limit of ed potato tissue (round 1), and resting spore suspensions detection. When using the tests for detection and iden- (round 2). Guidance in the EPPO standard on organisa- tification of S. endobioticum in resting spore suspen- tion of interlaboratory comparison studies (EPPO, sions, the Sendo PCR and Sendo TaqMan significantly 2014b) was found very helpful in setting up the TPS. outperformed the 1(D1) TaqMan for diagnostic sensi- Partners had to analyse 10 samples per TPS round. tivity and diagnostic specificity. For overall accuracy, When unclear or inconsistent results were obtained, the Sendo TaqMan significantly outperforms both the the analysis had to be repeated with a back-up sample Sendo PCR and 1(D1) TaqMan. Using the 1(D1) set. Datasets with incorrect results for the control sam- TaqMan for pathotype identification at low levels of the target proved to be difficult. Under the conditions ples were excluded from the analysis resulting in 13 datasets in round 1 and 14 (Sendo PCR), 13 (Sendo used in the TPS we would recommend to use caution TaqMan), and 7 (1(D1) TaqMan) datasets in round 2. when testing resting spore suspensions below 5000 Several TPS partners generated late Ct values for spores per sample. some of the healthy potato samples tested. A Ct cut-off For the determination of analytical specificity, sam- value was determined for the Sendo TaqMan to elimi- ples used were limited to different S. endobioticum nate late Ct values without introducing false negative pathotypes and healthy potato as no other Synchytrium results. These late Ct values could be the result of spp. were available to us. It is not likely that the symp- contamination or non-specific annealing of primers toms caused by potato wart disease are also induced by and probe. During the preparation of the TPS, late Ct closely related Synchytrium species as they are highly values in healthy potato tissue were observed only once. specialised for certain hosts. Wart disease symptoms After finalisation of the Euphresco Sendo project, re- could be confused with pseudo-wart: a proliferation of searchers at ILVO compared the Sendo TaqMan with a eyes that may be a physiological response, a varietal real-time PCR test targeting the small ribosomal subunit response, or could be induced by chemical factors. In (18S) described by Smith et al.(2014), which was not essence, potatoes with pseudo-wart are healthy potatoes. available at the time of the TPS setup, on extracts False non-pathotype 1(D1) results were obtained with obtained with zonal centrifugation (personal Swedish pathotype 1(D1) isolate (MB69) using the Eur J Plant Pathol (2018) 151:757–766 765 1(D1) TaqMan. Bonants et al.(2015) obtained similar in addressing this when new potato wart disease initia- results with some pathotype 1(D1) isolates originating tives are launched within this research framework. For from outside the Netherlands and Germany. The future diagnostic wart disease projects, recently pub- pathotype 1(D1) associated SNP that lies at the basis lished tests for molecular S. endobioticum detection of the 1(D1) TaqMan design was identified using Dutch (Smith et al., 2014), and genotype identification and German 1(D1) isolates. This means that for diag- (Gagnon et al. 2016) should be considered. nostic purposes, only pathotype 1(D1) positive results Acknowledgments We thank all colleagues for sharing experi- produced by the 1(D1) TaqMan can be used for molec- ences and providing feedback on the draft update of EPPO stan- ular 1(D1) identification. Strains identified as pathotype dard PM7/28 Synchytrium endobioticum. The work of NPPO-NL 1(D1) using a bioassay can produce non-pathotype staff members M.L Bruil-Dieters, J.P. Meffert, E. Metz-Verschure, 1(D1) results in the 1(D1) TaqMan. P.C.J. van Rijswick, E.J. van Veen and J.G.B. Voogd, to prepare TPS materials and perform homogeneity and stability experiments In addition, the 1(D1) TaqMan test was found diffi- is greatly recognised funding mechanism. cult to interpret as, apart from Ct values, participants had to identify pathotype 1(D1) and non-pathotype 1(D1) Compliance with Ethical Standards specific amplification plots as described by Bonants et al. (2015). This proved particularly challenging Conflict of Interest The authors declare that they have no for resting spore suspensions with low amount of conflict of interest. target. Some TPS partners indicated that, under their Human and Animal Rights This article does not contain any conditions, Ct values were as expected but that the studies with human participants or animals performed by any of obtained amplification plots were different com- the authors. This research project was performed within the pared to the expected reactions. The TPS organisers Euphresco II framework: Diagnostic methods for Synchytrium and some TPS partners also found slight differences endobioticum, especially for pathotype identification (SENDO), via a non-competitive. in the amplification plots for some S. endobioticum collection items compared to the results published by Bonants et al.. Real-time PCR machines, and in particular the ramp rates could have an influence on Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// the amplification efficiency and the corresponding creativecommons.org/licenses/by/4.0/), which permits unrestrict- shape of the real-time PCR amplification curves. ed use, distribution, and reproduction in any medium, provided This aspect has not been further investigated under you give appropriate credit to the original author(s) and the source, the TPS. The added value of the non-1(D1) reaction provide a link to the Creative Commons license, and indicate if changes were made. was questioned as it was found to be confusing. Im- proved user-friendliness of the 1(D1) TaqMan can be achieved by using the 1(D1) reaction without the non- References 1(D1) reaction in conjunction with one of the generic S. endobioticum tests. It is this combination we propose Baayen,R.P.,Cochius,G.,Hendriks,H.,Meffert,J.P.,Bakker,J., to include in the update of EPPO PM7/28. Bekker, M., van den Boogert, P. H. J. F., Stachewicz, H., & As goes for all potato wart disease studies, having van Leeuwen, G. C. M. (2006). History of potato wart disease access to sufficient and well characterised isolates cov- in Europe—a proposal for harmonisation in defining ering a broad geographical range is challenging because pathotypes. European Journal of Plant Pathology, 116,21–31. Bonants, P. J. M., van Gent-Pelzer, M. P. E., van Leeuwen, G. C. of the low outbreak frequency for this pest, the fact that M., & van der Lee, T. A. J. (2015). A real-time TaqMan PCR it is difficult to maintain in collections, and the different test to discriminate between pathotype 1 (D1) and non- pathotyping methods used in different laboratories. pathotype 1 (D1) isolates of Synchytrium endobioticum. However, having sufficient and well characterised iso- European Journal of Plant Pathology, 143,495–506. lates covering a broad geographical range is paramount EPPO. (2004). Diagnostic protocols for regulated pests, Synchytrium endobioticum. EPPO Bulletin, 34,213–218. for reliable development and validation of diagnostic EPPO. (2014a). EPPO Standards PM 7/98(2). Specific require- tests. The Synchytrium endobioticum community would ments for laboratories preparing accreditation for a plant pest strongly benefit from a centralised repository for collec- diagnostic activity. EPPO Bulletin, 44,117–147. tion material that maintains the material and keeps track EPPO. (2014b). EPPO Standards PM 7/122. PM 7/122 (1) of its Bgenealogy^. Euphresco partners could play a role Guidelines for the organization of interlaboratory 766 Eur J Plant Pathol (2018) 151:757–766 comparisons by plant pest diagnostic laboratories. EPPO Przetakiewicz, J. (2015a). The Viability of Winter Sporangia of Bulletin, 44,390–399. Synchytrium endobioticum (Schilb.) Perc. from Poland. American JournalofPotatoResearch,92,704–708. EPPO. (2017). PM 7/76 (4) Use of EPPO diagnostic protocols. Przetakiewicz, J. (2015b). First Report of New Pathotype 39(P1) EPPO Bulletin, 47,7–9. of Synchytrium endobioticum Causing Potato Wart Disease Gagnon, M. C., van der Lee, T. A., Bonants, P. J., Smith, D. S., Li, in Poland. Plant Disease, 99(2), 285. X., Lévesque, C. A., & Bilodeau, G. J. (2016). Development Smith, I. M., McNamara, D. G., Scott, P. 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Published: Feb 7, 2018