Dead or Alive, that Is the Question: Development and Assessment of Molecular Synchytrium endobioticum Viability Tests

Abstract

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, time-consuming and require specific expertise and/or 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 non-viable 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 genomic DNA of the target genes. We demonstrate the analytical sensitivity using synthetic constructs of the target mRNAs and 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. Biological relevance of mRNA detection was determined in the context of replicated Spieckermann bio-assays. Potential applications of the tests to facilitate collection management, assessment of the effects of treatments on presumed viability of S. endobioticum resting spores as well as the potential use in descheduling of previously infested plots, are discussed.