Azole resistance of
Aspergillus fumigatus
is a global problem. The major resistant mechanism is a cytochrome P
450
14-α sterol demethylase Cyp51A alteration such as mutation(s) in the gene and the acquisition of a tandem repeat in the promoter. Although other azole tolerances and resistant mechanisms such as
hmg1
(a 3-hydroxy-3-methylglutaryl-coenzyme-A reductase gene) mutation are known, few reports have described studies elucidating non-Cyp51A resistance mechanisms. This study explored genes contributing to azole tolerance in
A. fumigatus
by
in vitro
mutant selection with tebuconazole, an azole fungicide. After three-round selection, we obtained four isolates with low susceptibility to tebuconazole. These isolates also showed low susceptibility to itraconazole and voriconazole. Comparison of the genome sequences of the obtained isolates and the parental strain revealed a non-synonymous mutation in MfsD for a major facilitator superfamily protein (Afu1g11820, R337L mutation) in all isolates. Furthermore, non-synonymous mutations in AgcA for a mitochondrial inner membrane aspartate/glutamate transporter (Afu7g05220, E535Stop mutation), UbcD for a ubiquitin-conjugating enzyme E2 (Afu3g06030, T98K mutation), AbcJ for an ABC transporter (Afu3g12220, G297E mutation), and RttA for a putative protein
r
esponsible for
t
ebuconazole
t
olerance (Afu7g04740, A83T mutation), were found in at least one isolate. Disruption of the
agcA
gene led to decreased susceptibility to azoles. Reconstruction of the A83T point mutation in RttA led to decreased susceptibility to azoles. Reversion of T98K mutation to wild type in UbcD led to decreased susceptibility to azoles. These results suggest that these mutations contribute to lowered susceptibility to medical azoles and agricultural azole fungicides.