Molecular characterization and comparisons of potato wart (Synchytrium endobioticum) in historic collections to recent findings in Canada and the Netherlands

Abstract

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).

A target enrichment approach for enhanced recovery of Synchytrium endobioticum nuclear genome sequences

“…Resting spores were isolated from 200 g compost, which serve as inoculum for bio-assays, from the NPPO-NL S . endobioticum collection with a zonal centrifuge as described previously [ 5 ]. For the Canadian material, resting spores were collected in sieves, washed, and centrifuged, as described previously [ 8 – 10 ].…”

“… a SendoTrack-ID from [ 5 ] b C q values of the SSU TaqMan assay from [ 12 ], NA = not applicable or not done. …”

“…DNA was extracted from the European, Asian and Canadian samples following previously published protocols [ 5 ]. DNA concentrations were checked using a Qubit Flourometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA).…”

“…Sufficient sequencing coverage on the smaller mitochondrial genome of S . endobioticum was obtained in the previous study [ 5 ], but there was poor sequencing coverage of its nuclear genome. Sufficient coverage of the nuclear genome is essential for genome characterization, evaluation of nuclear encoded genes and their function, in particular effectors that are linked to pathotype identity [ 6 ].…”

Potato Wart Isolates from Europe and North America Form Distinct Clusters of Genetic Variation

“…To date, two complete genomes and partial genomic and transcriptomic datasets have been published for S. endobioticum, with assembly sizes of 21.48 and 23.21 Mb [ 17 ]. Along with the published nuclear genomes, the structure of the mitochondrial genome, a most likely maternally inherited linear molecule of 72.9 Kbp, has been published, and comparative analyses with the mt genomes of a number of genotypes have been performed [ 18 , 19 ]. These studies showed considerable variation between mitogenomes, with six different haplotypes identified, as well as putative admixtures in some of the populations studied.…”