Comparison of real‐time PCR tests for the detection of Synchytrium endobioticum resting spores in soil and their potential application in descheduling of previously infested plots

Plots infested with Synchytrium endobioticum, the causal agent of potato wart disease, are scheduled, resulting in prohibition cultivation and plants for planting. On account robust resting spores that present soils, plots remain scheduled 20 years. After this period, intensively sampled presence soil is determined by direct examination. However, method very time-consuming labour-intensive. In paper, validation data molecular detection its use to screen soils before examination reported. addition samples study, over 670 routine diagnostic were analysed tests parallel. Using an improved methodology, increased sensitivity was obtained relative results reported from interlaboratory comparison study 2018, namely 7 per sample instead 500 sample. Molecular screening those testing positive estimated reduce total hands-on time half when compared all samples. We recommend inclusion update EPPO PM 3/59 (3) 7/28(2), suggest subsamples a plot negative, no additional or bio-assays needed descheduling (i.e. releasing previously official control). Les parcelles infestés par l'agent de la galle verruqueuse pomme terre, sont réglementés, avec notamment interdiction culture terre et végétaux destinés à plantation. En raison présence dormantes robustes dans les sols infestés, restent réglementés pendant ans. Après ce lapse temps, échantillonnés manière intensive est évaluée examen du sol. Cette méthode cependant chronophage laborieuse. Cet article présente des données pour détection moléculaire le sol son utilisation un dépistage avant direct. plus échantillons l'étude validation, ont été analysés test en parallèle. A l’aide d’une méthodologie améliorée, une sensibilité accrue obtenue rapport aux résultats rapportés étude comparative inter-laboratoires réalisée savoir échantillon au lieu échantillon. Il estimé que l'examen seuls testés positifs permis réduire moitié temps manipulation tous échantillons. Nous recommandons d’inclure mise jour normes 7/28 (2) l'OEPP suggérons que, lorsque sous-échantillons d'une parcelle négatifs, aucun ou essai biologique complémentaire ne soit nécessaire déréglementation (c'est-à-dire, levée mesures lutte officielle sur précédemment infestées). Пoля, зapaжeнныe вoзбyдитeлeм paкa кapтoфeля, пoдлeжaт внeceнию в cпиcки зapaжённыx пoлeй, чтo пpивoдит к зaпpeтy нa выpaщивaниe ниx включaя pacтeний для пocaдки. Из-зa жизнecпocoбныx пoкoящиxcя cпop, кoтopыe пpиcyтcтвyют зapaжeннoй пoчвe, эти пoля ocтaютcя этиx cпиcкax тeчeниe лeт. Пo иcтeчeнии этoгo cpoкa пoляx интeнcивнo oтбиpaют oбpaзцы, и нaличиe cпop пoчвe oпpeдeляeтcя пyтeм пpямoгo иccлeдoвaния. Oднaкo этoт мeтoд oчeнь тpyдoeмкий зaнимaeт мнoгo вpeмeни. B cтaтьe пpeдcтaвлeны дaнныe пo вaлидaции мoлeкyляpнoгo мeтoдa oбнapyжeния eгo иcпoльзoвaниe cкpинингa пoчв пepeд oтчётoм o пpямoм иccлeдoвaнии. дoпoлнeниe oбpaзцaм вaлидaциoннoм иccлeдoвaнии, бoлee pyтинныx диaгнocтичecкиx oбpaзцoв были пapaллeльнo пpoaнaлизиpoвaны пpямым иccлeдoвaниeм мoлeкyляpными тecтaми. C пoмoщью ycoвepшeнcтвoвaннoй мeтoдики былa дocтигнyтa пoвышeннaя чyвcтвитeльнocть cpaвнeнию c peзyльтaтaми, пpeдcтaвлeнными мeжлaбopaтopнoм cpaвнитeльнoм иccлeдoвaнии 2018 гoдy, имeннo выявляeмocть oбpaзeц вмecтo oбpaзeц. Oцeнкa вcex пoчвы тex oбpaзцoв, дaли пoлoжитeльный peзyльтaт, пoкaзaлa coкpaщeниe oбщeгo вpeмeни paбoты двa paзa. Mы peкoмeндyeм включить мoлeкyляpнoe выявлeниe oбнoвлeнныe вepcии cтaндapтoв 7/28(2) EOКЗP пpeдлaгaeм, чтoбы пpи oтpицaтeльныx peзyльтaтax пoд-oбpaзцoв oднoгo нe тpeбoвaлocь дoпoлнитeльныx пpямыx иccлeдoвaний или биoaнaлизoв иcключeния из пepeчня пoлeй (тo ecть ocвoбoждeния paнee yчacткoв oт oфициaльнoй бopьбы). Potato disease important caused obligate biotrophic chytrid fungus endobioticum. infection susceptible varieties, typical cauliflower malformations formed, rendering crop unmarketable (Obidiegwu al., 2014). Complete yield loss has been as result infestation (Hampson, 1993). As part life cycle, produces can viable infectious decades (Przetakiewicz, 2015b). Isolates further characterized pathotypes based on ability form warts differential set cultivars (EPPO, 2017b). Currently 40 have described (Baayen 2006; Çakır 2009; Przetakiewicz, 2015a). there successful treatments control agents known eradicate 1988), prohibiting restricting resistant varieties buffer zones surrounding only ways prevent spread pathogen. Following initial pathogen, ‘scheduled’ prohibited that, determine if pathogen still present. Presence be after sieving washing combination field trials 2017a). Typically, performed first much quicker biological tests. When longer found present, may descheduled (Anonymous, 1969). Euphresco Sendo project, performance (TPS) compare several S. endobioticum warted tubers suspensions (Van Vossenberg 2018). The included endobioticum-specific conventional PCR den Boogert 2005), real-time (van Gent-Pelzer 2010), pathotype 1(D1) specific (Bonants 2015). DNeasy Plant Mini kit these well tissues, but analytical inadequate, limit Here we report two soils. These ITS2 Van al. (2010) 18S Smith (2014) filtration set-up CaCl2 purified Qiagen PowerSoil kit. annual surveys Dutch National Protection Organization (NPPO-NL) comparison, recommendations made plots. To effect DNA extraction kits tests, preliminary using three series freeze-dried (sample MB08, 2(G1)) healthy material (variety Eersteling) range 500, 50, 10, 5 1 extracted MB19, 18(T1)). materials below, processed (Qiagen, Hilden, Germany) PM7/28(2) vortex 10 min at maximum speed following manufacturer's instructions. Samples consisting 200 g compost used whole subsampled 25-g portions. (sub)samples stacked sieves mesh sizes 75 µm upper sieve 25 lower according standard PM7/28(2). sieved manually whereas 200-g subjected automated Analysette 18 heavy duty shaker (Fritsch, Idar-Oberstein, 200-µm, 71-µm 25-µm sieves. manual purification, 40–50 mL soil/spore suspension saturated solution obtained. For practical reasons, divided 50-mL tubes. Given uneven distribution spore (fragments) tubes not regarded duplicates. containing beaker fitted 20-µm nylon woven filter. connected vacuum pump (Figure S1). particular combination, cellulose nitrate filter provided had removed applying supernatant transferred without disrupting pellet, applied set-up. rinsed large amounts tap water collect filer. clean tube off water. centrifuged 5300 × concentrate pellet. pellet resuspended 60 µL C1 (Qiagen) 2-mL kit's PowerBeads. disrupted Mixer Mill (Retsch, Haan, 30 beats min. homogenate eluted 100 C6 immediately stored −20°C until use. Per round, (resting solution) negative isolation (molecular grade water) efficiency extraction. both methodology Standard pm7/28(2) followed. short, reaction mixes TaqMan Premix Ex Taq Master Mix (Takara Bio, Kusatsu, Japan) contained 250 nM primers ITS2F ITS2R, 83 probe 2. Genomic (3 µL) molecular-grade added reach final volume µL. Reaction Maxima qPCR master mix (Thermo Fisher, Waltham, USA) Se18S_RTF1 SE18S_RTR2, Se18S_TM1. (2 Apart generic plant cox1 separate reaction. internal Bio) COX F RW, SOL 1511T (Weller 2000; Mumford 2004). Amplification CFX96 touch (BioRad, Hercules, thermocyler program 95°C followed cycles 15 s 60°C Baseline threshold settings automatically amplification curves assessed visually. Where needed, baseline between 2 assay correct background signals corrected automatically. Tests considered they produced exponential Cq value below 40. Positive controls during each run. which detected, main types growing potatoes Netherlands clay, sandy ‘dal’ soil, latter being artificial topsoil subsoil removal intermediate peat) spiked 250, 50 5998981, 1(D1)) above. Results same number MGW inhibitory observed different types. extractions including solution. R2 combined selectivity general analysis variance (ANOVA) GenStat v21.1 (VSN International, Hemel Hempstead, United Kingdom) factors test, pellet) their interaction tested, means Bonferroni identify statistical significant groups. Soil taken regular 2019 2020 NPPO-NL study. 2019, 55 taken, 439 subsamples, 2020, 114 eight 226 100-g subsamples. 673 usual, meaning fraction floating counting chamber visually inspected under microscope. examination, content back original above subsequent test. survey samples, (composts ‘dal’, sand clay spores, n = 90) (n 51) molecularly. Technical duplicates tested controls, replicates detected. visual assessment define true agreement (PA), (NA), deviation (PD) (ND) determined, allowing calculation (PA/(PA + ND), specificity (NA/(NA PD) accuracy ((PA NA)/(PA NA PD ND)). criteria expressed percentages, providing insights into true-positive rate false-positive rate, overall significance differences two-sample binomial incorporated software. infected tuber expected qualitative methods (Table comparing TPS methods, (bead-beating) significantly (two-sided T-test, p < 0.05) outperformed (vortex) (meanPlant 19.5 ± 0.03, meanPowerSoil beads 19.9 0.93, 23.3 1.12). equally T-test: 0.51, 0.62). within TPS, i.e. meanVan 19.6 2.2. targeting tuber-derived mean 21.8 2.1. observed. managed detect more mini kit, could detected bead-beater, respectively considering 0.86) (mean 33.6 2.8) 33.4 2.9) contain little DNA, did produce except single case. Based results, bead-beating disruption selected PCRs determination characteristics. analysing series, five values 35.1 0.4 37.1 1.4 respectively. paired 0.001) 32.7 0.7 35.2 0.8 sample, four out S2). formula (LOD) 99.7% confidence level, LOD spiking numbers solutions free (general ANOVA: 0.566). Mean 29.0 30.2 34.6 34.8 1a). types, (ANOVA: 0.816) 1b). 29.3, 29.7 29.8 Again, extraction, higher matrices, 34.0, 34.9 S3). combining calculate R2, similar (R2ITS2 0.84 R218S 0.81). With average 1.8 than broader 2). Of 90 initially ‘positive’, 74 ranging ~30 000–50 000 estimated. 70 S4). remaining 16 ‘positive’ negative. 13 33.5 32.5 sensitivity, estimation 14 respective 51 ‘negative’, any Both majority One (Cqmean 34.5) 34.8) one 37.3). 169 survey, therefore 654 belonging subsample S5). ranged 33.3 37.5 32.1 39.4 representing <1 12 Direct new spores. 375 performed, 48% 35.7. Combining 1). perform (= rate), 97% corresponding 3%. (two-sample test: outperforms terms 95% 81% false-negative rates 5% 19% 96%, Overall, consistent results. 814 141 samples), 799 cases (consistency 98%). 80 PCRs, 0.2 1.26 lower. subset difference 0.133). it high signals. 44% 1624 reactions identified non-specific Whenever found, verification usually fairly straightforward. Typical summer winter material. Netherlands, planting least If so, period prolonged another 3 (60 0.33 ha) required reliably absence Furthermore, requires expertise distinguish structures resembling fungus. long-term experience, vast years will purification method. limited daily laborious consuming. sensitive pre-screen way save labour time, reliable outcomes. addition, mis-identification such conifer pollen. Only producing subsequently inspection. Recovery validation. those, (2010), TPS. fit introduction bead-beater (this study), PCR, set-up, rDNA (2014). typically consists (manual sieving) (automatic sieving), far recommended density gram partial preparing subjecting entire should avoided soil. disturbing efficient low-tech solution, benefit flotation This increases chance detecting buoyant. potential explain 3% diagnostics. low apparent ‘false positives’ ~1 likely represent missed might broken fragmented buoyant microscopist error, suggested 98% extracts consistently Nevertheless, fewer other possible filtration. Alternatively, occurred predominantly converted smaller potentially set, level attention microscopists raised, increasing detection. It noted taking lead From experiments need cut-off value, positive. fact diagnostics indicates matrix does introduce late values. fluorescent resulted reactions. inspection clearly signals, hampers user-friendliness believed length FAM-labelled oligo 32 bases long, inefficient quenching signal fluorophore quencher too apart. problem overcome introducing ZEN™ Double Quenched Probe. placed internally 9th 10th 5′ end probe, decreased fluorescence noise ratio (Wilson 2011). value. underlines external round laboratories access suspensions, problematic. reference fulfilled individual institutes collections, NPPO Polish Breeding Acclimatization Institute. coordinated approach would preferable, European Union Reference Laboratory (EURL) pests fungi oomycetes hosted French Agency Food, Environmental Occupational Health & Safety play role assigning delegated research collections EURL framework. viability RT-PCR mRNA currently developed Wageningen University, Canadian Food Inspection NPPO-NL. take strategy step further. DNA-based (fragmented) established. Instead labour-intensive error-prone establish expression genes viability. Moving towards allow Even though purpose descheduling. authors PM3/59 (2), Additionally, microscopically greatly greenhouse capacity affecting reliability strategy. Over last many people involved work leading manuscript. acknowledge contribution performing (parts of) workflow Specifically, like thank staff members Marcel Govaerts, Margriet Boerma (outsourced HLB, Wijster, Netherlands) Carin Helderman classical mycological analyses, Joël van der Loo, Lot Lubberts, Marlies Berg Hans Wolsink also Bert Waterink reviewing manuscript submission. Readers looking pdf paper copies consult html version online Supporting Information (Tables S1–S5). Please note: publisher responsible functionality supporting information supplied authors. Any queries (other missing content) directed author article.