Exploring the Molecular Mechanisms of Resistance to Prochloraz by Lasiodiplodia theobromae Isolated from Mango.

Abstract

Mango stem-end rot caused by Lasiodiplodia theobromae is a major postharvest disease in China. Prochloraz is commonly used for disease control in mango orchards and in storage. However, prochloraz resistance has been detected in L. theobromae. This study aimed to explore the underlying mechanisms responsible for prochloraz resistance in L. theobromae. The results show that no point mutation in the target gene LtCYP51 of the prochloraz-resistant L. theobromae strain was detected, but the expression was upregulated significantly. Additionally, the full-length sequences of the cytochrome P450 gene CYP55A3 were successfully amplified and identified from L. theobromae, and the qRT-PCR results confirm that CYP55A3 was significantly upregulated after treatment with prochloraz. The knockout mutant of the CYP55A3 presented significantly lower gene expression levels than the wild-type strain HL02, with a 16.67-fold reduction, but a 1.34-fold reduction in P450 activities and a 1.72-fold increase in the accumulation of prochloraz in the mycelia. Treatment with the P450 enzyme inhibitor significantly synergized with the prochloraz toxicity. The wild-type strain was highly resistant to pyraclostrobin and carbendazim; similarly, the sensitivity of the knockout mutant to pyraclostrobin and carbendazim also notably increased. There was no significant difference between the wild-type strain and the gene-complemented strain. The homology model and molecular docking analysis provide evidence that prochloraz interacts with the protein structure of CYP55A3. These findings suggest that the overexpression of the target gene LtCYP51 and the detoxification gene CYP55A3 were involved in the molecular mechanisms of resistance to prochloraz by L. theobromae.