Multiple Pro-197 substitutions in the acetolactate synthase of rigid ryegrass (Lolium rigidum) and their impact on chlorsulfuron activity and plant growth

The evaluation of 21 Lolium rigidum accessions for chlorsulfuron resistance in a whole-plant response experiment indicated that 10 accessions were highly resistant (R) to chlorsulfuron, three partially resistant (r) and seven susceptible (S) to this herbicide. The excellent control of the r accessions in the presence of terbufos provided strong evidence for the existence of herbicide metabolism resistance due to cytochrome P450 enzymes. The sequence of 34 L. rigidum plants from the 10 R accessions revealed that all mutant ALS alleles encoded an amino acid replacement at codon 197, which resulted in the substitution of Pro-197 by Ala, Arg, Gln, Leu, or Ser and probably His or Val. Most of the R accessions revealed more than one mutation and among them the Pro-197 by Ala was the most common, whereas twelve out of the 34 R sequenced individual plants were homozygous. The sequence of 12 and 9 L. rigidum plants from the respective four S and three r accessions did not indicate any ALS mutation at the codon 197. ALS activity assays confirmed the whole-plant response experiments and the ALS gene sequence results. The resistance ratios (R/S) for the R accessions ranged from 36 to 620 showing that ALS gene mutations resulted in less susceptible to chlorsulfuron ALS enzyme. The R/S values for the r accessions were similar to the S-control accession. The growth rate evaluation of four R and four S L. rigidum accessions in the absence of competition indicated that the R accessions had similar growth pattern to that of S accessions, suggesting that the resistance-endowing ALS mutations did not result in detectable resistance fitness cost. ► Ten out of 21 rigid ryegrass accessions were resistant to chlorsulfuron. ► In ten accessions resistance was due to altered target-site at Pro-197. ► In three accessions resistance was due to chlorsulfuron metabolism. ► Point mutations resulted in less susceptible to chlorsulfuron ALS. ► Target-site resistance did not result in any detectable fitness cost.