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

Abstract

Stem-end rot (SER) caused by Lasiodiplodia theobromae is an important disease of mango in China. Demethylation inhibitor (DMI) fungicide are widely used for diseases control in mango orchards. The baseline sensitivity to difenoconazole of 138 isolates collected in the field in 2019 from mango were established by the mycelial growth rate method. The cross-resistance to six site-specific fungicides with different modes of action were investigated using 20 isolates randomly selected. The possible reasons for L. theobromae resistance to difenoconazole were preliminarily determined through gene sequence alignment and quantitative real-time PCR analysis. The results showed that the EC50 values of 138 L. theobromae isolates to difenoconazole ranged from 0.01 to 13.72 µg/ml. The frequency of difenoconazole sensitivity formed a normal distribution curve when the outliers were excluded. Difenoconazole showed positive cross-resistance only with the DMI tebuconazole, but not with non-DMI carbendazim, pyraclostrobin, fludioxonil, bromothalonil, or iprodione. Some multifungicide-resistant isolates of L. theobromae were found. Two amino acid substitutions (E209k and G207A) were found in CYP51 protein, but they were not likely related to the resistance phenotype. There was no alteration in promoter region of the CYP51 gene. However, difenoconazole significantly increased the expression of the CYP51 gene in the resistant isolates when compared to the susceptible isolates. This study is important references to explore resistance mechanism. These results are vital to make effective mango diseases management strategies in order to avoid the development of further resistance.