Molecular mechanism of Glutathione S-Transferase involved in resistance to multiple fungicide in Lasiodiplodia theobromae.

Stem-end rot caused by Lasiodiplodia theobromae is a significant postharvest disease in mango, with the pathogen developing resistance to multiple fungicides (MDR) in China. This study investigates the role of GST genes in L. theobromae fungicide resistance. The GST genes related to MDR was screened and identified through bioinformatic and transcriptomic, qRT-PCR, and genetic manipulation. The SY302 isolate showed resistance to carbendazim, difenoconazole and pyraclostrobin. Synergistic effects of the GST inhibitor diethylmaleate (DEM) with difenoconazole or pyraclostrobin resulted in enhanced fungicide toxicity (SR values from 18.46 to 24.00). GST activity was significantly higher in the resistant isolate after treatment with difenoconazole and pyraclostrobin. RNA-Seq identified 23 differentially expressed GST genes, with LtGST4 and LtGST9 significantly upregulated in the resistant isolate and considered as the important candidate genes. These genes were amplified and bioinformatic analysis revealed that LtGST4 belongs to the Ure2p subfamily and LtGST9 to the GTT1 subfamily. Gene knockout and overexpression experiments showed that knocking out the LtGST4 and LtGST9 genes of the resistant isolate resulted in a notable enhance in fungicides sensitivity, reducing the L. theobromae resistance to three fungicides. Conversely, overexpressing of the LtGST4 and LtGST9 genes of the sensitive isolate led to obviously decreased fungicides sensitivity, enhancing the L. theobromae tolerance to three fungicides. This study provides experimental evidence that LtGST4 and LtGST9 contribute to MDR in L. theobromae by mediating fungicide detoxification. The finding enrich the understanding of metabolic gene mediated fungicide resistance and offering insights for future plant pathogen control strategies.