Target gene mutations endowed cross-resistance to acetolactate synthase-inhibiting herbicides in wild Brassica juncea

Wild Brassica juncea is a troublesome weed that infests wheat fields in China. Two suspected wild B. juncea populations (19–5 and 19–6) resistant to acetolactate synthase (ALS) inhibitors were collected from wheat fields in China. To clarify their resistance profiles and resistance mechanism, the resistance levels of populations 19–5 and 19–6 to ALS-inhibiting herbicides and their underlying target-site resistance mechanism were investigated. The results showed that the 19–5 population exhibited resistance to tribenuron-methyl, pyrithiobac‑sodium and florasulam, while the 19–6 population was resistant to tribenuron-methyl, pyrithiobac‑sodium, imazethapyr and florasulam. Using the homologous cloning method, two ALS genes were identified in wild B. juncea, with one gene (ALS1) encoding 652 amino acids and the other (ALS2) encoding 655 amino acids. Pro-197-Arg mutation on ALS2 and Trp-574-Leu mutation on ALS1, together with the combination of these two mutations in a single plant, were observed in both 19–5 and 19–6 populations. ALS2 enzymes carrying the Pro-197-Arg mutation were cross-resistant to tribenuron-methyl, pyrithiobac‑sodium, imazerthapyr and florasulam, with resistance index (RI) values of 6.23, 32.81, 7.97 and 1162.50, respectively. Similarly, ALS1 enzymes with Trp-574-leu substitutions also displayed high resistance to these four herbicides (RI values ranging from 132.61 to 3375.00). In addition, the combination of Pro-197-Arg (ALS2) and Trp-574-Leu (ALS1) mutations increased the resistance level of the ALS enzyme to ALS inhibitors, with its RI values 3.83–214.19, 6.88–37.34, 1.91–31.82 and 2.03–5.90-fold higher than a single mutation for tribenuron-methyl, pyrithiobac‑sodium, imazerthapyr and florasulam, respectively. Collectively, Pro-197-Arg mutation on ALS2, Trp-574-Leu mutation on ALS1 and the combination of Pro-197-Arg (ALS2) and Trp-574-Leu (ALS1) mutations in wild B. juncea could endow broad-spectrum resistance to ALS inhibitors, which might provide guides for establishing effective strategies to prevent or delay such resistance evolution in this weed.