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Amaranthus hybridus  is one of the main weed species in Córdoba, Argentina. Until recently, this weed was effectively controlled with recurrent use of glyphosate. However, a population exhibiting multiple resistance (MR2) to glyphosate and imazamox appeared in a glyphosate resistant (GR) soybean field, with levels of resistance up to 93 and 38-fold higher to glyphosate and imazamox, respectively compared to the susceptible (S) population. In addition to imidazolinones, MR2 plants showed high resistance levels to sulfonylamino-carbonyl (thio) benzoates and moderate resistance to sulfonylureas and triazolopyrimidines. Multiple amino acid substitutions were found in both target genes, acetolactate synthase (ALS) and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), responsible for conferring high herbicides resistance levels in this  A. hybridus  population. In the case of  EPSPS , the triple amino acid substitution TAP-IVS was found. In addition, MR2 plants also showed increased  EPSPS  gene expression compared to susceptible plants. A Ser653Asn substitution was found in the  ALS  sequence of MR2, explaining the pattern of cross-resistance to the ALS-inhibitor herbicide families found at the ALS enzyme activity level. No other mutations were found in other conserved domains of the  ALS  gene. This is the first report worldwide of the target site resistance mechanisms to glyphosate and ALS inhibitors in multiple herbicide resistance  Amaranthus hybridus .

Multiple mutations in the EPSPS and ALS genes of Amaranthus hybridus underlie resistance to glyphosate and ALS inhibitors