Synchytrium endobioticum (Schilb.) Perc. is a chytrid fungus causing potato wart disease and is one of the most important quarantine diseases on cultivated potato. Infected host tissues develop warts rendering the crop unmarketable. Resting spores, that can remain viable and infectious for decades, are formed in warted tissues and are released into the surrounding soil when host tissue decays.To better understand the pathogen’s diversity and to potentially uncover pathways of migrations and introduction events, molecular characterization was performed on the historical S. endobioticum resting spore collection of the Dutch National Plant Protection Organization. Mitochondrial genomes were assembled and annotated, and four novel structural variants were identified from these materials with intronic presence-absence variation in cox1 or cob genes and structural variation in the dpoB – TIR region. Several fungal isolates were shown to contain mixtures of structural variants. We analyzed the mitogenomic sequences obtained from recent potato wart disease findings in Canada and the Netherlands in the context of the historical materials and found that fungal isolates from the new Dutch outbreak contained a specific mixture of mitogenomic variants previously not observed in the Netherlands. Based on the mitogenomic profile, pathotype 38(Nevşehir) was suspected which was later verified with the Spieckermann bioassay. To further facilitate dissemination of data and interactive visual analytics we created a public Nextstrain webpage with S. endobioticum mitogenomic sequences and associated metadata on their geographic origin, pathotype identity and (mixture) of mitogenomic variants (https://nextstrain.nrcnvwa.nl/Sendo).

Plots infested with Synchytrium endobioticum, the causal agent of potato wart disease, are scheduled, resulting in prohibition of potato cultivation of and cultivation of plants for planting. On account of robust resting spores that are present in infested soils, plots remain scheduled for 20 years. After this period, plots are intensively sampled and the presence of resting spores in soil is determined by direct examination. However, this method is very time-consuming and labour-intensive.In this paper, validation data for the molecular detection of resting spores in soil and its use to screen soils before direct examination are reported. In addition to the samples in the validation study, over 670 routine diagnostic samples were analysed by direct examination and molecular tests in parallel. Using an improved methodology, increased sensitivity was obtained relative to results reported from an interlaboratory comparison study in 2018, namely 7 spores per sample instead of 500 spores per sample. Molecular screening of the soil samples and direct examination of those samples testing positive was estimated to reduce the total hands-on time by half when compared to direct examination of all samples. We recommend inclusion of molecular detection in the update of EPPO PM 3/59 (3) and PM 7/28(2), and suggest that when all subsamples of a plot are negative, no additional direct examination or bio-assays are needed for descheduling (i.e. for releasing previously infested plots from official control). Comparaison de tests PCR en temps réel pour la détection de spores dormantes de Synchytrium endobioticum dans le sol et leur application potentielle pour la déréglementation des parcelles précédemment infestésLes parcelles infestés par Synchytrium endobioticum, l'agent causal de la galle

Synchytrium endobioticum (Schilb.) Perc. is a chytrid fungus causing potato wart disease and is one of the most important quarantine diseases on cultivated potato. Infected host tissues develop warts rendering the crop unmarketable. Resting spores that can remain viable and infectious for decades are formed in warted tissues which are released into the surrounding soil when the host tissue decays. To better understand the pathogen’s diversity and to potentially uncover pathways of migrations and introduction events, molecular characterization was performed on the historical S. endobioticum resting spore collection of the Dutch National Plant Protection Organization. Mitochondrial genomes were assembled and annotated, and four novel structural variants were identified from these materials with intronic presence-absence variation in cox1 or cob genes and structural variation in the dpoB – TIR region. Several fungal isolates were shown to contain mixtures of structural variants. We analyzed the mitogenomic sequences obtained from recent potato wart disease findings in Canada and the Netherlands in the context of the historical materials and found that fungal isolates from the new Dutch outbreak contained a specific mixture of mitogenomic variants previously not observed in the Netherlands. Based on the mitogenomic profile, pathotype 38(Nevşehir) was suspected which was later verified by pathotyping. To further facilitate dissemination of data and interactive visual analytics we created a public Nextstrain webpage with S. endobioticum mitogenomic sequences and associated metadata on their geographic origin, pathotype identity and (mixture) of mitogenomic variants ( https://nextstrain.nrcnvwa.nl/Sendo ).