Exploration of the potential of high throughput DNA sequencing to quantify fungicide resistance in populations of Zymoseptoria tritici

Abstract

Septoria tritici blotch (STB) caused by Zymoseptoria tritici is the most damaging foliar disease of wheat in Northern Europe, causing up to 50% yield loss each year. Common STB control strategies include the application of fungicides. However, intensive fungicide use has driven a selection for resistant isolates and some fungicides have as a result become less effective, or in extreme cases totally ineffective. Moreover, in the last decades there has been a reduction in the availability of fungicides to control this disease due to regulation processes and slow development of new active ingredients, which creates a threat for the EU wheat production. There are three fungicidal modes of action that are commonly used for STB: multi-site inhibitors (MSI), succinate dehydrogenase inhibitors (SDHIs) and demethylation inhibitors (DMIs, usually referred as azoles). Traditionally, screening of pathogen populations require the isolation and culturing of the pathogen, which is slow and expensive. Here, a different strategy based on sequencing using Oxford Nanopore Technologies (ONT) was explored to understand the haplotype and incidence of resistance within populations of Z. tritici. The method was optimized to enable rapid sequencing of fungicide target genes for isolates, populations, and mock communities. The aim was to facilitate a full, rapid assessment of existing resistance status leading to more informed decision-making regarding fungicide use by farmers, such as minimising the risk of further resistant strains. To understand the genetic mechanisms that underly fungicide resistance, the sensitivity of 49 isolates of Z. tritici to four widely used azoles was tested. The level of resistance was determined in culture and linked to the fungicide resistance mechanisms, which included mutations on CYP51, insertions within its promoter region and overexpression of the ABC and MFS transporters. Findings showed that the analysis of all three mechanisms, is required for a better understanding of the phenotype. Moreover, the haplotype of CYP51 and the four SDH subunits was tested to determine how the population structure changed throughout the season after spraying single active ingredients.