Resistance to ALS-inhibiting herbicides in common ragweed, foxtail and horseweed

Acetolactate synthase (ALS) is a key enzyme in the biosynthesis of the branched-chain amino acids valine, leucine, and isoleucine. Five commercialized classes of ALS-inhibiting herbicides, sulfonylureas (SUs), imidazolinones (IMIs), triazolopyrimidines (TPs), pyrimidinylthiobenzoates (PTBs), and sulfonylaminocarbonyltriazolinones (SCTs) are known to inhibit ALS. However, weed resistance to ALS inhibitors evolved quickly, with the first resistant prickly lettuce observed only 5 years after the first use of SU herbicides. The overall objective of this study was to gain insights into the molecular mechanisms of resistance to ALS inhibitors in common ragweed (Ambrosia artemisiifolia), foxtail (Setaria spp.) and horseweed (Conyza canadensis ).;In most cases, resistance to ALS-inhibiting herbicides is due to an altered target site with reduced sensitivity to the herbicides. ALS enzyme activity assay results confirmed that resistance in several biotypes used in this study were target-site based and biotypes had different cross-resistance patterns among ALS-inhibitor classes at the enzyme level. Since different groups of herbicides have slightly different binding sites on the acetolactate synthase molecule, these results indicated that these biotypes possessed different mechanisms of ALS-inhibitor resistance.;Amino acid substitutions that confer resistance to ALS inhibitors in weeds have been reported at six different sites (Ala122, Pro 197, Ala205, Asp376, Tryp574 and Ser 653), and each site is located within a highly conserved region of the ALS enzyme. Based on PCR amplification of specific alleles (PASA) and herbicide efficacy data, it was concluded that tryptophan to leucine substitution at position 574 was the predominant basis for resistance to cloransulam in common ragweed. However, this mutation could not fully account for resistance in several populations, which displayed resistance to imazamox but not to cloransulam. Entire ALS gene sequence data indicated that valine to alanine substitution at position 205 was also a resistance-conferring mutation in common ragweed populations.;Polyploidy among foxtail species complicated the study of ALS inhibitor resistance because the number of ALS genes varied with the level of ploidy. Chromosome counting and flow cytometry were used to detect the ploidy level for each species. The results indicated that green foxtail was diploid (2X=18) whereas giant and yellow foxtail were tetraploid (4X=36). Consistent with this, Southern blot analysis revealed that green foxtail had one copy of ALS, and both giant and yellow foxtail had two copies of ALS. Substitution at position 653 was associated with IMI resistance in green and yellow foxtail. Both W574L and S653N substitutions were identified in giant foxtail.;The entire coding sequence of the ALS gene from horseweed was amplified and sequenced from sensitive and resistant horseweed biotypes using polymerase chain reaction primers created from Helianthus annuus and Xanthium strumarium ALS gene sequences. The amino acid sequences were deduced and compared to identify amino acid polymorphisms conferring ALS inhibitor resistance. Substitutions at amino acid positions 197 and 376 were identified.;Based on the results and mechanisms identified from the above three weeds, PCR-based molecular markers have been used in this study to investigate the evolution of cloransulam resistance in common ragweed, which appeared in the same year as cloransulam was commercialized. The results revealed that resistance to cloransulam in common ragweed evolved in response to selection by ALS inhibitors other than cloransulam. Also, resistance-conferring ALS alleles were used to develop a marker to assess the genotype of horseweed at codons encoding amino acid position 197. In this way we have found an effective approach to confirm the hybridization of horseweed biotypes by applying a PCR-based molecular marker. This marker is being used in ongoing studies to investigate the inheritance of glyphosate resistance in horseweed.