Identification of resistance to multiple herbicides and metabolic resistance to penoxsulam in barnyardgrass (Echinochloa crus-galli).

Barnyardgrass (Echinochloa crus-galli) is a problematic weed in rice production, and resistance to multiple herbicides threatens sustainable management. The resistant (R) population JZWD lacks ALS target-site mutations for penoxsulam, indicating metabolic resistance. Whole-plant assays revealed that JZWD exhibited moderate resistance to penoxsulam, glufosinate ammonium and haloxyfop-P, and low resistance to quinclorac, glyphosate and halauxifen-methyl, while it was susceptible to quizalofop-p-ethyl. The synergistic effects of malathion confirmed the involvement of P450 in penoxsulam resistance. Transcriptome sequencing (RNA-Seq) of untreated and penoxsulam-treated resistant and susceptible plants revealed 25 candidate genes belonging to major detoxification enzyme families (12 cytochrome P450 monooxygenases, 3 glutathione S-transferases, 3 uridine diphosphate-dependent glycos-yltransferases, 3 ATP-binding cassette transporters and 4 peroxidase). Four core genes (CYP72A616-like, UGT83A1, CYP97B2 and PODSPC4-1) were significantly upregulated in at least two resistance-related comparisons. KEGG enrichment revealed primary metabolism pathways enriched among the upregulated genes. Technical qRT-PCR validation of the same RNA samples confirmed the RNA-Seq results for most genes. Independent biological samples revealed that CYP72A616-like was constitutively overexpressed (35.87-fold) in the R population, whereas UGT83A1, CYP97B2 and PODSPC4-1 were strongly induced by penoxsulam (8.75 to 334.92-fold). Two additional P450s (CYP71C42 and CYP734A1) were also upregulated. Among the three additional resistant populations, four representative P450 genes showed variable upregulation patterns under untreated conditions (14.79 to 160.19-fold), and all three populations were strongly induced by penoxsulam (16.25 to 945.07-fold). These results suggest that metabolic resistance mediated by P450s and other detoxification enzymes underlies multiple herbicide resistance in E. crus-galli, providing targets for resistance monitoring and sustainable weed management.