Difenoconazole Resistance Shift in Botrytis cinerea From Tomato in China Associated With Inducible Expression of CYP51

Abstract

Gray mold caused by Botrytis cinerea is one of the most important diseases in tomato. It can be controlled effectively by demethylation inhibitor (DMI) fungicides, but their resistant status is unclear after long-term use in the field. Baseline sensitivity to difenoconazole of 142 B. cinerea isolates from China with no history of DMI usage was characterized, with a mean EC50 of 0.97 ± 0.50 μg/mL. EC50 values to difenoconazole of another 248 isolates collected in 2011 and 2016 ranged from 0.04 to 11.99 μg/mL, and the frequency of difenoconazole sensitivity formed a non-normal distribution curve. Detached fruit studies revealed that isolates with EC50 values of ~6.00 μg/ml were not controlled effectively. The mean EC50 of the resistant isolates changed from 6.74 to 8.65 μg/mL between 2011 and 2016. Positive cross-resistance was only observed between difenoconazole and two DMIs. One dual resistant and one triple resistant isolates were found among the difenoconazole-resistant isolates collected in 2016, associated with point mutations in corresponding target proteins of the fungicides azoxystrobin and fludioxonil. This indicated that B. cinerea not only showed higher difenoconazole resistance levels but gradually changed from single to multiple fungicide resistance over time. No amino acid variation was found in the CYP51 protein. In the absence of difenoconazole, the relative expression of CYP51 was not significantly different in sensitive and resistant isolates. Induced expression of CYP51 is an important determinant of DMI resistance in B. cinerea from tomato. However, nucleotide variants found in the upstream region had no association with the fungicide resistance phenotype. These results will be helpful for the management of B. cinerea in the field.

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

“…In addition, no cross-resistance was discovered in this investigation between DMI and non-DMI fungicides. This is consistent with the reported results of Botrytis cinerea and Colletotrichum gloeosporioides resistance to DMIs 16 , 37 . In the experiment of cross resistance, we found that there were a small number of isolates with multifungicide resistance (MFR).…”

“…According to the report of Rallos, overexpression of CYP51 was associated with the presence of the Y136F mutant genotype 45 . However, the underlying mechanisms of CYP51 overexpression are not known for in the field DMI-resistant subpopulations of Puccinia triticina 43 , Sclerotinia homoeocarpa 51 , Pyrenophora teres 17 , Colletotrichum gloeosporioides 16 , and Botrytis cinerea 37 . We tried to clone and sequence analysis the promoter of LtCYP51 in this study.…”

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

“…In addition, no cross-resistance was discovered in this investigation between DMIs and non-DMIs fungicides. This matches reports of Botrytis cinerea and Colletotrichum gloeosporioides resistance to DMIs 16,37 . The commonly used site-speci c fungicides (such as carbendazim and azoxystrobin) gave bad control effect against L. theobromae due to the development of resistant isolates.…”

“…For example, a 199-bp sequence duplication was discovered at the promoter of CYP51 of Penicillium digitatum 45 . However, underlying mechanisms of CYP51 overexpression are not known for in the eld DMI-resistant subpopulations of Puccinia triticina 39 , Sclerotinia homoeocarpa 46 , Pyrenophora teres 17 , Colletotrichum gloeosporioides 16 , Botrytis cinerea 37 . We tried to clone and sequence analysis the promoter of LtCYP51 in this study.…”

Multiple Mutations and Overexpression in the CYP51A and B Genes Lead to Decreased Sensitivity of Venturia effusa to Tebuconazole

“…Mutations in the CYP51 gene are a common cause of resistance to DMIs and have been found in several other pathogens [36,41,42]. Overexpression of the CYP51 genes is also commonly found in resistant isolates of various pathogens [43][44][45]. We anticipated either mutations or overexpression of the CYP51 gene to be the cause of the resistance, and we found mutations and overexpression to be present in DMI-resistant isolates of V. effusa.…”