Molecular Basis for the Resistance to ALS-Inhibiting Herbicides in Amaranthus palmeri

LARRAN ALVARO; LIEBER LUCAS; LORENZETTI FLORENCIA; MONTERO BULACIO NICOLÁS; TUESCA DANIEL; PERMINGEAT HUGO RAÚL

Foz de Iguazú

Congreso; 11th International Congress of Plant Molecular Biology (IPMB); 2015

International Society for Plant Molecular Biology

Herbicide resistant weeds are a serious problem worldwide, particularly affecting crops systems in USA, Brazil and Argentina. Amaranthus palmeri is a native species from the south-west of North America and was first detected in Argentina during 2011 in the south-west of the Córdoba province. Recently, a population with suspected cross-resistance to acteolactate synthase (ALS) inhibiting herbicides (R biotype) was found in the town of Vicuña Mackenna. In most reported cases, resistance to ALS inhibitors is caused by point mutations in the als gene causing amino acid substitutions within highly conserved regions of the protein sequence1.A. palmeri plants of the resistant biotype and a susceptible one collected from Tucumán province (S byotipe) were subjected to in vivo dose-response assays with three herbicides containing active ingredients of different families: sulfonylureas (SUs), imidazolinones (IMIs) and triazolopyrimidines (TPs). Subsequently, genomic DNA extraction was performed and als gene was amplified with primers designed from als sequences of related species published in database2,3. Amplification products were purified, ligated to pGEM®-T easy vector and cloned into DH5α E. coli strain. Plasmids were purified from individual clones. Insert incorporation was checked by both colony-PCR and enzymatic digestion. Eight positive-recombinant DNA molecules from each byotipe were sequenced through Macrogen?s service, employing primers that were designed to accurately cover the entire sequence of the gene. Using the bioinformatic software Unipro Ugene v1.11.2, gene sequences were compared with those of A. thaliana and related species of the genus Amaranthus, and were screened for mutations that could account for the resistance observed. Also, identity matrices between nucleotide and amino acid sequences were built.R biotype showed a strong resistance to the three active ingredients tested: chlorimuron-ethyl (SU), diclosulam (TP) and imazetapir (IMI), when compared to the susceptible population (S). Resistance levels reached values corresponding to thirty-two times the dose applied to field. Although nucleotide sequence comparison of als genes from R and S biotypes revealed a 99% of identity, one nucleotide difference resulted in R biotype having a tryptophan to leucine amino acid substitution at position 574, as numbered relative to the protein sequence of A. thaliana.