Generating single spore isolates of Phakopsora pachyrhizi for a better understanding of fungicide resistance mechanisms

摘要

The generation of single spore isolates is usually the first approach to characterize plant fungal pathogens with respect to their specific resistance mechanisms to fungicides. The isolation procedure can be time-consuming and can carry the risk of contamination, but for fungal species with saprophytic growth (e.g., Zymoseptoria tritici) it is simple to perform in most cases. However, for biotrophic fungi such as Phakopsora pachyrhizi the situation is more difficult. This may be the reason why all published studies on fungicide resistance mechanisms of this fungus have been conducted with populations or monouredinial isolates. Monouredinial isolates, in contrast to single spore isolates, are generated from several spores of one uredinium, and thus, it is not ensured that these isolates are clones and therefore genetically identical. Since only dikaryotic uredospores of P. pachyrhizi have been found in the field so far, the mechanisms of fungicide resistance are more complex than for other pathogens. Therefore, the use of clones as a tool for further characterization is mandatory. We established a reliable method to generate single spore isolates of P. pachyrhizi and demonstrated that the isolates are clonal. For this verification, single spore isolates were used in detached leaf tests (DLTs) and treated with two demethylation inhibitors (DMIs) with different selection pattern. These isolates had different frequency values of three relevant mutations in CYP51. Additionally, a population was included in the DLT that represented a mixture of isolates with the respective CYP51 mutations under the detection limit (< 5). In contrast to the population in which target site mutations were selected after DMI treatments in an expected manner, the single spore isolates showed no change in their mutation frequencies on untreated or treated leaves. This indicates that these isolates are genetically identical and thus valuable for further elucidation of the complex resistance mechanisms, especially against DMIs, in this fungal species.