Abstract
Lanosterol 14-α demethylase is a key enzyme intermediating the biosynthesis of ergosterol in fungi, and the target of azole fungicides. Studies have suggested that Leptosphaeria maculans and L. biglobosa, the causal agents of phoma stem canker on oilseed rape, differ in their sensitivity to some azoles, which could be driving pathogen frequency change in crops. Here we used CYP51 protein modelling and heterologous expression to determine whether there are interspecific differences at the target-site level. Moreover, we provide an example of intrinsic sensitivity differences exhibited by both Leptosphaeria spp. in vitro and in planta. Comparison of homologous protein models identified highly conserved residues, particularly at the azole binding site, and heterologous expression of LmCYP51B and LbCYP51B, with fungicide sensitivity testing of the transformants, suggests that both proteins are similarly sensitive to azole fungicides flusilazole, prothioconazole-desthio and tebuconazole. Fungicide sensitivity testing on isolates shows that they sometimes have a minor difference in sensitivity in vitro and in planta. These results suggest that azole fungicides remain a useful component of integrated phoma stem canker control in the UK due to their effectiveness on both Leptosphaeria spp. Other factors, such as varietal resistance or climate, may be driving observed frequency changes between species.
Highlights
Antifungal demethylation inhibitors (DMIs), e.g. triazoles and imidazoles, target lanosterol 14α-demethylase (CYP51, erg11), a member of the cytochrome P450 superfamily and key regulatory enzyme in the ergosterol biosynthetic pathway[1,2]
Using predictive homology modelling supported by heterologous expression in a Saccharomyces cerevisiae mutant, we have investigated if CYP51 structural differences play a role in fungicide-sensitivity differences previously reported between Leptosphaeria maculans and L. biglobosa populations
Heterologous expression of lanosterol 14α-demethylase (CYP51B) from Leptosphaeria maculans (LmCYP51B) and L. biglobosa (LbCYP51B), together with subsequent fungicide sensitivity assays on the yeast transformants, show that LmCYP51B and LbCYP51B interact with azole fungicides tebuconazole, flusilazole and prothioconazole-desthio. These observations support the homology modelling predictions that LmCYP51B and LbCYP51B share a high level of structural similarity, at the azole-binding site
Summary
Antifungal demethylation inhibitors (DMIs), e.g. triazoles and imidazoles (azoles), target lanosterol 14α-demethylase (CYP51, erg11), a member of the cytochrome P450 superfamily and key regulatory enzyme in the ergosterol biosynthetic pathway[1,2]. Ergosterol is an essential structural component of the fungal plasma membrane and is required to maintain membrane integrity, fluidity and permeability. Azoles are used to control fungal pathogens in both clinical and agricultural situations. A wide range of azole fungicides have been used to control fungal plant pathogens since their inception in the 1970s6. Agricultural azole fungicides are used as a component of fungicide programmes (often in mixtures) to control fungal diseases, including phoma stem canker of winter oilseed rape (Brassica napus) caused by coexisting fungal plant pathogens Leptosphaeria maculans and L. biglobosa[9]. Differences in sensitivity between coexisting plant pathogens can affect population structure[13] and it has been suggested that lower azole sensitivity could explain the recent increase in L. biglobosa incidence in some UK locations[14]
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