A portable, nanopore-based genotyping platform for near real-time detection of Puccinia graminis f. sp. tritici lineages and fungicide sensitivity

Background: Fungal plant disease outbreaks are increasing in both scale and frequency, posing severe threats to agroecosystem stability, native biodiversity and food security. Among these, the notorious wheat stem rust fungus, Puccinia graminis f.sp. tritici (Pgt), has threatened wheat production since the earliest days of agriculture. New Pgt strains continue to emerge and quickly spread over vast distances through the airborne dispersal of asexual urediniospores, triggering extensive disease outbreaks as these exotic Pgt strains often overcome resistance in dominant crop varieties of newly affected regions. This highlights the urgent need for a point-of-care, real-time Pgt genotyping platform to facilitate early detection of emerging Pgt strains. Results: In this study, we developed a simple amplicon-based re-sequencing platform for rapid genotyping of Pgt isolates. This system is built around a core set of 276 Pgt genes that we found are highly polymorphic between Pgt isolates and showed that the sequence of these genes alone could be used to accurately type Pgt strains to particular lineages. We also developed a simplistic DNA preparation method and an automated bioinformatic pipeline, to enable these Pgt gene markers to be sequenced and analysed rapidly using the MinION nanopore sequencing device. This approach successfully enabled the typing of Pgt strains within approximately 48 h of collecting Pgt-infected wheat samples, even in resource-limited locations in Kenya and Ethiopia. In addition, we incorporated monitoring capabilities for sequence variations in Pgt genes that encode targets of the azole and succinate dehydrogenase inhibitor fungicides, enabling real-time tracking of potential shifts in fungicide sensitivity. Conclusion: The newly developed Pgt Mobile And Real-time, PLant disEase (MARPLE) diagnostics platform we established, now allows precise typing of individual Pgt strains while simultaneously tracking changes in fungicide sensitivity, providing an early warning system for potential indicators of changes in the Pgt population and emerging fungicide resistance. Further integration of this Pgt MARPLE diagnostics platform into national surveillance programmes will support more informed management decisions and timely responses to Pgt disease outbreaks, helping reduce the devastating crop losses currently caused by this ‘cereal killer’.