Two Point Mutations on CYP51 Combined With Induced Expression of the Target Gene Appeared to Mediate Pyrisoxazole Resistance in Botrytis cinerea

Abstract

Botrytis cinerea is a destructive plant pathogenic ascomycete that causes serious pre-and post-harvest losses worldwide. The novel sterol 14α-demethylase inhibitor (DMI) pyrisoxazole was recently registered for the control of tomato gray mold caused by B. cinerea in China. Baseline sensitivity of 110 B. cinerea isolates collected from nine provinces in China to pyrisoxazole was demonstrated, with a mean EC 50 of 0.057 ± 0.029 µg/ml. Eleven stable mutants resistant to pyrisoxazole were generated via UV irradiation (RU-mutants) and spontaneous selection (RS-mutants) of conidia. The efficacy of pyrisoxazole against the resistant mutants was significantly lower than that of the sensitive isolates. Most of the pyrisoxazole-resistant mutants were less fit than the sensitive isolates, with reduced sporulation, conidia germination, sclerotium production, and pathogenicity, which was confirmed by the competitive ability test. Positive cross-resistance was only observed between pyrisoxazole and the DMIs tebuconazole and prochloraz, but not between pyrisoxazole and non-DMIs iprodione, procymidone, diethofencarb, fluazinam, pyrimethanil, or fludioxonil. A two-point mutation, at G476S and K104E in the RU-mutants, and a one point mutation, M231T, in the RS-mutants, were detected in the CYP51 protein of the resistant mutants. When exposed to pyrisoxazole, the induced expression level of CYP51 increased in the resistant isolates as compared to sensitive ones. Molecular docking suggested that G476S and M231T mutations both led to the loss of electrostatic interactions between CYP51 and pyrisoxazole, while no change was found with the K104E mutation. Thus, two point mutations on CYP51 protein combined with induced expression of its target gene appeared to mediate the pyrisoxazole resistance of B cinerea.

Molecular Characterization and Overexpression of CYP51 gene of Difenoconazole Resistance in Lasiodiplodia Theobromae Field Isolates

“…Mango diseases are widely controlled using site-speci c systemic fungicides in almost all mangogrowing regions in the world. The detection of fungicide resistance is a crucial step in monitoring and regulating the spread of resistance in the eld 20 . Among systemic fungicides, MBC fungicides are inhibitors of tubulin biosynthesis and impede cell division and inhibited mycelial growth 21 .…”

“…DMI fungicides are classi ed as a medium risk for resistance development by the Fungicide Resistance Action Committee 26 . However, DMIs resistance has been found in a variety of phytopathogenic fungi 17,16,20 . The resistance mechanisms of DMIs have been reported to be diverse, with the following being the primary mechanisms: (I) point mutations in the target gene 14demethylase (CYP51) [27][28][29] ; (II) CYP51 gene overexpression [30][31][32][33][34]16 ; (III) Overexpression of e ux proteins 35,36 .…”

“…simultaneously in some DMIs-resistant isolates of plant pathogens 17,42,20 . An increase in DMI fungicide application dosages would not improve their e cacy in the case of mutation.…”

Molecular characterization and overexpression of the difenoconazole resistance gene CYP51 in Lasiodiplodia theobromae field isolates

“…Mango diseases are widely controlled using site-specific systemic fungicides in almost all mango-growing regions in the world. The detection of fungicide resistance is a crucial step in monitoring and regulating the spread of resistance in the field 20 . DMI fungicides are classified as a medium risk for resistance development by the Fungicide Resistance Action Committee 21 .…”

“…DMIs fungicides were more favoured by orchardist due to their specific mode of action and broad anti-fungi spectrum at present. However, DMI resistance has been found in a variety of phytopathogenic fungi 16 , 17 , 20 . The resistance mechanisms of DMIs have been reported to be diverse: (I) point mutations in the target gene 14α-demethylase (CYP51) 22 – 24 ; (II) CYP51 gene overexpression 16 , 25 – 29 ; and (III) overexpression of efflux proteins 30 , 31 .…”