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

Mango stem-end rot caused by <i>Lasiodiplodia theobromae</i> 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 <i>L. theobromae</i>. This study aimed to explore the underlying mechanisms responsible for prochloraz resistance in <i>L. theobromae</i>. The results show that no point mutation in the target gene <i>LtCYP51</i> of the prochloraz-resistant <i>L. theobromae</i> strain was detected, but the expression was upregulated significantly. Additionally, the full-length sequences of the cytochrome P450 gene <i>CYP55A3</i> were successfully amplified and identified from <i>L. theobromae</i>, and the qRT-PCR results confirm that <i>CYP55A3</i> was significantly upregulated after treatment with prochloraz. The knockout mutant of the <i>CYP55A3</i> 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 <i>LtCYP51</i> and the detoxification gene <i>CYP55A3</i> were involved in the molecular mechanisms of resistance to prochloraz by <i>L. theobromae</i>.