Characterisation of Mycosphaerella graminicola isolates with reduced azole sensitivities (PhD)

Fungicide activity against Mycosphaerella graminicola, the causal agent of Septoria leaf blotch (SLB) on wheat has declined over recent years. The overall aim of this study was to characterise UK M. graminicola field isolates and to identify the molecular mechanisms that result in reduced azole (triazole and imidazole) sensitivity.

M. graminicola isolates from treated and untreated UK field populations in 2004 had significantly lower epoxiconazole sensitivities compared to isolates from 1993. The prevalence of alterations in eburicol 14α-demethylase (CYP51), the azole target site, was assessed within the1993 and 2004 (?) isolates. This confirmed that substitution I381V correlates with slightly reduced epoxiconazole sensitivity. Cross-resistance occurred within the triazole and the imidazole groups, but not between groups. Fourteen compounds were assessed for their ability to increase epoxiconazole efficacy against isolates with reduced azole sensitivity. A formula, developed to calculate synergistic interactions, identified synergism between trifluoperazine or fluphenazine and epoxiconazole. In future this could be used to identify synergistic compounds to aid fungicide longevity.

Expression of 16 M. graminicola transporter genes (including 11 novel genes) was studied in several isolates, both in planta and in vitro. None of these genes were overexpressed to the extent expected to cause a reduction in epoxiconazole sensitivity. However, expression of a selection of these genes was induced by epoxiconazole in a resistant isolate. In planta, it was possible to identify genes involved in infection of the leaf but not those involved in reducing fungicide sensitivity. 

Biochemical techniques were used to analyse differences in sterol content between sensitive isolates and isolates with reduced epoxiconazole sensitivities. Using sterol content data, it was possible to assess overall CYP51 activity, and identify the impact of individual mutations on activity. Some alterations clearly reduce CYP51 activity, whereas others probably compensate for a loss of activity. These data suggest that adaptation of M. graminicola to azole fungicide use, particularly evolution of the CYP51 target enzyme, is an ongoing dynamic process in modern populations of the fungus.