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, pyrithiobac­sodium and florasulam, while the 19-6 population was resistant to tribenuron-methyl, pyrithiobac­sodium, 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, pyrithiobac­sodium, 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, pyrithiobac­sodium, 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.

Variation in mutations providing resistance to acetohydroxyacid synthase inhibitors in Cyperus difformis in China.

Cyperus difformis has evolved resistance to pyrazosulfuron-ethyl and other acetohydroxyacid synthase (AHAS) inhibitors in paddy fields in China. To understand the distribution of resistance and the mutations involved, 38 populations collected were from 7 provinces and compared. Application of pyrazosulfuron-ethyl at 30 g a.i. ha-1 identified 16 populations that survived, demonstrating resistance to this herbicide. Two exons of 498 and 1428 bp in length and a 1228-1233-bp intron of AHAS were cloned by genome walking, and three pairs of primers were designed to amplify eight conserved regions in this gene. In the 16 resistant (R) populations, five different types of mutations in the conserved region of the AHAS gene were identified: Pro-197-Ser, Pro-197-Arg, Pro-197-Leu, Asp-376-Glu, and Trp-574-Leu. Three R populations, YX15-22, YX12-10 and YX15-38, were chosen for in vitro AHAS activity assays, and the results showed that AHAS from YX15-22 carrying the Pro-197 mutation was insensitive to pyrazosulfuron-ethyl (resistance index (RI) = 310.0) and penoxsulam (RI = 10.0), whereas the enzyme from YX12--10 and YX15-38 was insensitive to pyrazosulfuron-ethyl, penoxsulam, imazapic and bispyribac­sodium (RI values ranging from 4.3 to 4462.0). AHAS inhibitor cross-resistance bioassays showed that YX12-10 and YX15-38 had cross-resistance to all of the tested herbicides (RI values ranging from 5.8 to 3321.9), while the YX15-22 population only had resistance to pyrazosulfuron-ethyl (RI = 827.4) and penoxsulam (RI = 6.6). This study clarified the distribution of resistant C. difformis in China and the different cross-resistance patterns given by various mutation types of AHAS.