Demethylase Inhibitor Fungicide Resistance in Pyrenophora teres f. sp. teres Associated with Target Site Modification and Inducible Overexpression of Cyp51

Abstract

Pyrenophora teres f. sp. teres is the cause of net form of net blotch (NFNB), an economically important foliar disease in barley (Hordeum vulgare). Net and spot forms of net blotch are widely controlled using site-specific systemic fungicides. Although resistance to succinate dehydrogenase inhibitors and quinone outside inhibitors has been addressed before in net blotches, mechanisms controlling demethylation inhibitor resistance have not yet been reported at the molecular level. Here we report the isolation of strains of NFNB in Australia since 2013 resistant to a range of demethylase inhibitor fungicides. Cyp51A:KO103-A1, an allele with the mutation F489L, corresponding to the archetype F495I in Aspergillus fumigatus, was only present in resistant strains and was correlated with resistance factors to various demethylase inhibitors ranging from 1.1 for epoxiconazole to 31.7 for prochloraz. Structural in silico modeling of the sensitive and resistant CYP51A proteins docked with different demethylase inhibitor fungicides showed how the interaction of F489L within the heme cavity produced a localized constriction of the region adjacent to the docking site that is predicted to result in lower binding affinities. Resistant strains also displayed enhanced induced expression of the two Cyp51A paralogs and of Cyp51B genes. While Cyp51B was found to be constitutively expressed in the absence of fungicide, Cyp51A was only detected at extremely low levels. Under fungicide induction, expression of Cyp51B, Cyp51A2, and Cyp51A1 was shown to be 1.6-, 3,- and 5.3-fold higher, respectively in the resistant isolate compared to the wild type. These increased levels of expression were not supported by changes in the promoters of any of the three genes. The implications of these findings on demethylase inhibitor activity will require current net blotch management strategies to be reconsidered in order to avoid the development of further resistance and preserve the lifespan of fungicides in use.

Induced expression of CYP51 associated with difenoconazole resistance in the pathogenic Alternaria sect. on potato in China

“…In this case, an increase in the application doses of DMI fungicide would not improve their efficacy. Many point mutations have been reported in CYP51 of various plant pathogens, among which the mutation at AA136 conferring the change from tyrosine to phenylalanine (Y136F) is the most common . However, only a mutation at AA511 conferring the change of arginine to tryptophan (R511W) was found in one of our resistant isolates, indicating other mechanisms were responsible for the development of DMI resistance.…”

A new mechanism for reduced sensitivity to demethylation‐inhibitor fungicides in the fungal banana black Sigatoka pathogen Pseudocercospora fijiensis

“…PfCYP51 substitutions Y136F, A313G, A381G, Y461D, Y463D, Y463H and Y463N were found in the present study, in accordance with previous observations for P. fijiensis exposed to propiconazole (Cañas‐Gutiérrez et al ., ), as well as in exposure to other azoles in Zymoseptoria tritici , Candida albicans , Pyrenophora teres f. sp . teres and Aspergillus fumigatus (Akins and Sobel, ; Cools and Fraaije, ; Mair et al ., ; Mellado et al ., ). Unexpectedly, we identified a 100‐bp insertion in the Pfcyp51 promoter region in addition to the coding region mutations in most P. fijiensis isolates from the Cartagena population.…”

Metyltetraprole, a novel putative complex III inhibitor, targets known QoI‐resistant strains of Zymoseptoria tritici and Pyrenophora teres

“…DMI resistance is associated with point mutations or overexpression of the enzyme encoded by CYP51 and efflux pump overexpression . Resistance to SDHIs is also caused by mutations in the Sdh genes encoding the subunits of complex II in the respiratory chain .…”