Complaints of control failures with acetolactate synthase (ALS)- and protoporphyrinogen oxidase (PPO)-inhibiting herbicides on redroot pigweed (Amaranthus retroflexus L.) were reported in conventional soybean [Glycine max (L.) Merr.] fields in North Carolina. Greenhouse dose–response assays confirmed that the Camden County and Pasquotank County populations were less sensitive to ALS- and PPO-inhibiting herbicides compared with susceptible A. retroflexus populations, suggesting the evolution of resistance to these herbicides. Sanger sequencing of target genes determined the Camden County population carried a Trp-574-Leu mutation in the ALS gene and an Arg-98-Gly mutation in the PPX2 gene, while the Pasquotank County population carried a His-197-Pro mutation in the ALS gene (first documentation of the mutation in the Amaranthus genus), but no mutation was detected in the PPX2 gene. Single-nucleotide polymorphism (SNP) genotyping assays were developed to enable efficient screening of future control failures in order to limit the spread of these herbicide-resistant populations. In addition, preliminary testing of these assays revealed the three mutations were ubiquitous in the respective populations. These two populations represent the first confirmed cases of PPO-inhibiting herbicide-resistant A. retroflexus in the United States, as well as the first confirmed cases of this particular herbicide-resistance profile in A. retroflexus inhabiting North America. While no mutation was found in the PPX2 gene of the Pasquotank County population, we suggest that this population has evolved resistance to PPO-inhibiting herbicides, but the mechanism of resistance is to be determined.

Soybean is a major crop cultivated in Brazil, and acetolactate synthase (ALS)-inhibiting herbicides are widely used to control weeds in this crop. The continuous use of these ALS-inhibiting herbicides has led to the evolution of herbicide-resistant weeds worldwide. Greater beggarticks is a polyploid species and one of the most troublesome weeds in soybean production since the discovery of ALS-resistant biotypes in 1996. To confirm and characterize the resistance of greater beggarticks to ALS inhibitors, whole-plant bioassays and enzyme experiments were conducted. To investigate the molecular basis of resistance in greater beggarticks the ALS gene was sequenced and compared between susceptible and resistant biotypes. Our results confirmed that greater beggarticks is resistant to ALS inhibitors and also indicated it possesses at least three isoforms of the ALS gene. Analysis of the nucleotide and deduced amino acid sequences among the isoforms and between the biotypes indicated that a single point mutation, G–T, in one ALS isoform from the resistant biotype resulted in an amino acid substitution, Trp574Leu. Two additional substitutions were observed, Phe116Leu and Phe149Ser, in a second isoform of the resistant biotype, which were not yet reported in any other herbicide-resistant ALS gene; thus, their role in conferring herbicide resistance is not yet ascertained. This is the first report of ALS mutations in an important, herbicide-resistant weed species from Brazil.

The molecular basis of resistance to tribenuron-methyl, an acetolactate synthase (ALS)–inhibiting herbicide was investigated in four resistant (R) and three susceptible (S) flixweed populations. The resistance level in the R populations was assessed in whole-plant pot experiments in a greenhouse, and resistance indices ranged from 723 to 1422. The ALS genes of the three S populations and four R populations were cloned and sequenced, and the full coding sequence of the ALS gene of flixweed was 2,004 bp. The sequences of the ALS genes of the three S populations collected from Shaanxi, Gansu, and Tianjin were identical. Comparison of the ALS gene sequences of the S and R populations with Arabidopsis revealed that proline at position 197 of the ALS gene was substituted by leucine in R population SSX-2, by alanine in R population SSX-3, and by serine in R populations TJ-2 and GS-2. In another study of two R flixweed populations from Hebei and Shaanxi, resistance was also related to mutation at position 197 of the ALS gene. Both studies confirmed tribenuron-methyl resistance in flixweed in China, with the resistance mechanism being conferred by specific ALS point mutations at amino acid position 197.

Reports arose from major Chinese wheat production regions that flixweed was not controlled by tribenuron after the herbicide was continuously used for several years. Flixweed seeds were collected from wheat fields that had been treated with tribenuron repeatedly over 3 to 15 yr or from road sides and remote hills that had never received tribenuron in Jiangsu, Hebei, Shanxi, Sichuan, Shandong, Shaanxi, and Henan provinces, and Tianjin and Beijing metropolises in China. The response of various biotypes to tribenuron was determined by whole plant experiments in the greenhouse. The experiments demonstrated that 11 of 32 flixweed biotypes were susceptible to tribenuron. The remaining 21 biotypes expressed moderate to high levels of tribenuron resistance with resistance indices ranging from 4 to > 1,500. DNA sequence analysis of acetolactate synthase (ALS) genes of selected biotypes 6, 7, 27, and 29 revealed a point mutation at position 197 of the ALS gene, as numbered relative to the protein sequence of Arabidopsis, where proline was substituted by leucine in biotype 7 and by threonine in biotype 29. These mutations are known to confer resistance to ALS-inhibiting herbicides and are responsible for the high resistance of these biotypes to tribenuron. The results also indicate that tribenuron resistance in flixweed is widespread in China and management programs to control these resistant populations are warranted.

Multiple-herbicide resistance represents an added weed management challenge to growers as it can considerably reduce their options for weed control. The widespread nature of triazine resistance in Ontario coupled with the more recent appearance of resistance to ALS inhibitors in Amaranthus species warranted documenting biotypes with multiple resistance. A collection of Powell amaranth and redroot pigweed biotypes that had previously been characterized for resistance to ALS inhibitors was therefore screened with atrazine. Dose–response analysis with atrazine and imazethapyr was also conducted. High-level resistance to imazethapyr and atrazine was determined in a Powell amaranth biotype from Perth County, Ontario. This biotype had a > 1,860-fold and 109-fold resistance to atrazine and imazethapyr, respectively. Sequence analysis was conducted for the psbA and ALS genes that code for the target sites of the triazines and imidazolinones, respectively. A mutation in the psbA gene was identified that coded for an amino acid substitution of glycine for serine at residue 264 of the D1 protein. This mutation is the most likely cause for triazine resistance in this biotype. Similarly, a nucleotide substitution was identified that codes for threonine in place of serine at position 652 of the ALS protein. This mutation in the ALS gene has only been observed previously in laboratory-selected mutants of arabidopsis and tobacco and is known to endow resistance to imidazolinones in plants. It is concluded that multiple resistance in this Powell amaranth biotype is due to the presence of altered target sites for triazine and imidazolinone herbicides.

Populations of water starwort, a winter annual or biennial weed in the pink family (Caryophyllaceae), can no longer be controlled by tribenuron following successive use of this herbicide over several years. Whole-plant bioassays have established that the resistant water starwort populations JS17, JS08, JS16, and JS07 showed high-level (from 203-fold to 565-fold) resistance to tribenuron. In vitro acetolactate synthase (ALS) assays revealed that resistance was due to reduced sensitivity of the ALS enzyme to tribenuron. The half-maximal inhibitory concentration (I50) values for JS17, JS08, JS16, and JS07 were 72, 71, 70, and 76 times greater, respectively, than were those of the susceptible population JS24. This altered ALS sensitivity in the resistant populations was due to a mutation in the ALS gene resulting in a Pro197 to Ser substitution (JS17, JS08, and JS16) and a Pro197 to Leu substitution (JS07). This study established the first documented case, to our knowledge, of evolved tribenuron resistance in water starwort and concluded that the molecular basis of resistance is due, at least in part, to a target-site modification at Pro197 in the ALS gene.