During the last year we`ve been working with several A. quitensis biotypes wich were not controlled with usual dosis of glyphosate in agricultural fields.

We performed greenhouse studies comparing them with a known susceptible biotipe and we found a very high level of resistance in three of the suspected biotypes.

The dose response experiments were carried on using a wide range of doses from 34 g. a.e/ha up to 17280 g e.a/ha.  In all the suspected biotypes we registered surviving plants even at the highest dose tested. 

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Contributors: Daniel Tuesca, Juan Carlos Papa; Sergio Morichetti and Nicolás Montero Bulacio.

A triple target-site mutation confers high levels of resistance to glyphosate in an Amaranthus quitensis population from Argentina   Sarah-Jane Hutchings^a, Eddie McIndoe^a, Ryan Carlin^b, Wenjin Yu^b, Anushka Howell^a, Weining Gu^b, Wenling Wang^b, Mike Langford^a, Jo Mattocks^a and Shiv-Shankar Kaundun^{a   a} Syngenta, Jealott’s Hill International Research Centre, RG42 6EY Bracknell, UK ^b Syngenta, Research Triangle Park, NC USA   E:mail: sarah-jane.hutchings@syngenta.com

Resistance to glyphosate has evolved and is quickly spreading in Amaranthus quitensis due to excessive use of the EPSPS-inhibiting herbicide in Argentinian soybean production systems.  Here, we confirmed resistance to glyphosate and determined the mechanism involved in an A. quitensis population (AMAQU-R) from Santa Fe Province, Argentina. AMAQU-R plants survived glyphosate rates as high as 48 kg ai/ha, whereas individuals from the sensitive populations (AMAQU-S1 and AMAQU-S2) were killed at 750 g ai/ha or below. Differential glyphosate uptake, movement or metabolism was not associated with resistance in AMAQU-R and only a small average relative increase (1.94) in EPSPS gene copy number/expression was detected in AMAQU-R.  Three linked EPSPS mutations were identified around the binding site of glyphosate in AMAQU-R. These comprised T102I and P106S amino acid substitutions documented to confer high levels of glyphosate resistance, as well as a novel A103V target-site mutation. A 3.5-fold more tolerance to glyphosate was observed for the TIAVPS mutant compared to the double TIPS strain following heterologous gene expression and enzyme analysis. The triple glyphosate resistance mutations may explain the rapid spread of A. quitensis in Argentina and could be a real threat for neighbouring countries under similar glyphosate selection pressure.

A triple mutation in the epsps gene is responsable of high levels of resistance to glyphosate in Amaranthus hybridus from Argentina

Valeria Perotti, Alvaro Larran, Valeria Palmieri y Hugo Permingeat.

Traditionally, the smooth pigweed (Amaranthus hybridus, previously known as A. quitensis) has been one of the most problematic weeds in the summer crops of our country, not only for its physiology (extraordinary growth and reproduction rates) but also for the appearance of subpopulations with multiple herbicide resistance. The finding of a subpopulation resistant to ALS inhibitors and to glyphosate in Canals (Córdoba) gave us the material to fulfill an objective: to elucidate the molecular basis of the resistance, in this case, to glyphosate. After the confirmation of the agronomic resistance by dose-response assays (where LD₅₀ and GR₅₀ showed to be higher than 17 Kg ha^-1), the accumulation of shikimate in leaf disks of single plants was determined. Thus, we obtained values from 4 to 7 ng of shikimate per microliter for sensitive plants (S), and close to 0 for resistant plants (R), suggesting a target-site mechanism. Subsequently, the sequencing of the epsps gene region enclosing the Proline 106 (a position which mutated is responsible for glyphosate resistance in different weeds) was carried out. The PCR products of 12 R plants and 2 S plants were sequenced, finding the so-called TIPS double mutation (corresponding to the substitutions T102I and P106S) in 8 R plants, confirming a target-site mechanism.  A third mutation was found to cause an alanine-valine substitution at position 103 of the amino acid sequence (taking as reference the EPSPS sequence of maize). This replacement represents a very interesting finding from a biotechnological point of view, since it could be involved in the unusually high glyphosate resistance levels observed in this subpopulation. Further research is necessary to confirm this hypothesis. Finally, the comparative estimation of the epsps gene copy number was performed using the als gene as a reference. These results showed that the subpopulation of Canals has an average increase of 3.6 times relative to the subpopulation S. Thus, the present work allowed to elucidate for the first time the mechanism endowing glyphosate resistance in A. hybridus species. 

Key words: smooth pigweed, EPSPS, triple mutation, Amaranthus quitensis