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Potato wart disease is caused by the obligate fungal pathogen Synchytrium endobioticum. DNA extraction from compost, purified spores and crude wart tissue derived from tuber galls of infected potatoes often results in low S. endobioticum DNA concentration or highly contaminated with DNA coming from other microorganisms and the potato host. Therefore, Illumina sequencing of these samples generally results in suboptimal recovery of the nuclear genome sequences of S. endobioticum. A hybridization-based target enrichment protocol was developed to strongly enhance the recovery of S. endobioticum DNA while off-target organisms DNA remains uncaptured. The design strategy involved creating a set of 180,000 molecular baits targeting both gene and non-gene regions of S. endobioticum. The baits were applied to whole genome amplified DNA samples of various S. endobioticum pathotypes (races) in compost, from purified spores and crude wart tissue samples. This was followed by Illumina sequencing and bioinformatic analyses. Compared to non-enriched samples, target enriched samples: 1) showed a significant increase in the proportion of sequenced bases mapped to the S. endobioticum nuclear genome, especially for crude wart tissue samples; 2) yielded sequencing data with higher and better nuclear genome coverage; 3) biased genome assembly towards S. endobioticum sequences, yielding smaller assembly sizes but higher representation of putative S. endobioticum contigs; 4) showed an increase in the number of S. endobioticum genes detected in the genome assemblies. Our hybridization-based target enrichment protocol offers a valuable tool for enhancing genome sequencing and NGS-based molecular detection of S. endobioticum, especially in difficult samples.

Potato wart disease caused by the obligate biotrophic fungus Synchytrium endobioticum is a devastating disease that can result in significant crop losses. Resting spores of this pathogen can remain viable and infectious in soil for decades. The detection of viable resting spores using conventional methods such as bioassays and direct microscopic examination are challenging and time-consuming and require specific expertise and facilities. Molecular methods, such as real-time PCR, have been shown to be effective in detecting the presence of S. endobioticum DNA in soil samples but cannot differentiate between viable and nonviable spores. In this paper, we present three novel mRNA-based molecular tests to potentially detect viable S. endobioticum resting spores. The tests are specific to the transcribed mRNA and do not detect the genomic DNA of the target genes. We demonstrate the analytical sensitivity using synthetic constructs of the target mRNAs. The tests were found to be able to repeatedly detect 10 target copies per reaction. Soils and waste of potato processing industries free from S. endobioticum were used to assess the exclusivity of the tests. The biological relevance of mRNA detection was determined in the context of replicated bioassays. Applications of the tests to facilitate collection management, assessment of the effects of treatments on presumed viability of S. endobioticum resting spores, and the potential use in descheduling of previously infested plots are discussed.