Use of loop-mediated isothermal amplification assays to detect azole-insensitive CYP51- overexpressing strains of Zymoseptoria tritici

Septoria leaf blotch is currently controlled by programmed applications of multisite inhibitors (e.g. chlorothalonil), azoles (e.g. epoxiconazole and prothioconazole) and a new generation of Succinate Dehydrogenase Inhibitors (e.g. bixafen, isopyrazam and fluxapyroxad). Azole fungicides have been used for three decades but their efficacy has eroded over time due to the evolution of azole insensitive strains carrying alterations in the sterol 14α-demethylase (CYP51) target protein. Because of the importance of azoles as a mixing partner of SDHIs continued monitoring of azole sensitivity shifts is paramount. Recently, we have reported a new mechanism, a 120 bp insertion in the CYP51 promoter which is linked with 10- to 40-fold CYP51 overexpression. Other isolates carry promoter variants based on a larger insert of 868 bp but the impact of this insert on the regulation of CYP51 expression remains unclear. Here we present the development and application of loop-mediated isothermal amplification (LAMP) assays for rapid, on the spot detection of CYP51 promoter inserts in Zymoseptoria tritici (Mycosphaerella graminicola) isolates carrying CYP51 variants [L50S, S188N, I381V, ΔY459/G460 & N513K] and [L50S, S188N, A379G, I381V, ΔY459/G460 & N513K].