Aspectos agronómicos, biológicos y moleculares de biotipos resistentes al herbicida glifosato

Ensayos de laboratorio e invernadero se llevaron a cabo para investigar las diferencias en susceptibilidad entre tres especies del género Conyza introducidas como malas hierbas en España: Conyza bonariensis, Conyza canadensis y Conyza sumatrensis. El material vegetal se obtuvo de semillas recolectadas en los cultivos de olivar y cítricos en el sur de España, sin registro ninguno de aplicaciones previas del herbicida glifosato. Las curvas dosis-respuesta mostraron diferentes valores de ED50: 2.9, 15.7 y 34.9 g ia (ingrediente activo) ha-1, respectivamente para C. sumatrensis, C. bonariensis y C. canadensis en el estadío de roseta (6- 8 hojas). Diferencias significativas se encontraron entre las tres especies en la retención foliar de herbicida, así como también en el ángulo de contacto. El orden de acuerdo a la retención foliar del herbicida fue C. sumatrensis > C. bonariensis > C. canadensis, mientras que los valores medios del ángulo de contacto fueron 59.2, 65.5 y 72.9º, respectivamente. No existieron diferencias significativas entre las tres especies de acuerdo a la absorción foliar de 14C-glifosato (los valores encontrados fueron desde 37.4% para C. canadensis a 52.4% para C. sumatrensis) el orden entre las especies fue el mismo que la retención foliar del herbicida. La cantidad de radiactividad translocada de las hojas tratadas fue menor en C. canadensis comparada con las otras dos especies (C. sumatrensis > C. bonariensis > C. canadensis). Entre todos los resultados de los parámetros estudiados, se ha identificado una susceptibilidad diferencial al herbicida glifosato entre las especies de Conyza. Existió una acumulación de ácido shiquímico después de la aplicación de glifosato a la dosis de 200 g ia ha-1. Sin embargo, C. canadensis mostró una menor cantidad de ácido shiquímico respecto a las otras dos especies a 168 h después del tratamiento. En C. bonariensis, se realizaron estudios eninvernadero para determinar la susceptibilidad al herbicida glifosato en tres estadíos diferentes de crecimiento: roseta, elongación del tallo (10-15 cm de altura) y floración. El menor ED50 obtenido fue durante el estadío de roseta (15.7 g ia ha-1), comparado con la elongación del tallo (86.6 g ia ha-1), obteniendo el mayor ED50 durante el estadío de floración (117.5 g ia ha-1); las plantas en un estadío temprano de crecimiento retuvieron una mayor cantidad de glifosato. Los resultados están en concordancia con observaciones en campo donde las plantas más jóvenes son más susceptibles al herbicida glifosato

Overexpression of Acetyl CoA Carboxylase 1 and 3 (ACCase1 and ACCase3), and CYP81A21 were related to cyhalofop resistance in a barnyardgrass accession from Arkansas

Barnyardgrass [Echinochloa crus-galli (L.) P. Beauv.] is the most difficult-to-control weed species of rice production systems worldwide. It has evolved resistance to different herbicide sites of action, including the acetyl-CoA carboxylase (ACCase)-inhibiting herbicides. Target-site mutations conferring resistance to ACCase-inhibiting herbicides are well documented; however, the role of the different ACCase genes in conferring resistance to cyhalofop-p-butyl (cyhalofop), an ACCase-inhibiting herbicide, remains poorly understood. This research assessed the contribution of gene amplification and expression of ACCase genes in a cyhalofop-resistant barnyardgrass accession. Additionally, the expression of glutathione-S-transferases (GSTs) and cytochrome P450 monooxygenases (P450s) genes as possible contributors to resistance to cyhalofop were investigated. Results demonstrated that ACCase gene amplification does not contribute to cyhalofop resistance. However, ACCase1 and ACCase3 were found to be overexpressed in the cyhalofop-resistant barnyardgrass accession. At 24 h after cyhalofop treatment, an overexpression of 2.0- and 2.8-fold was detected in ACCase1 and ACCase3, respectively. In addition, CYP81A21 (a P450 gene) was found to be 2.5-fold overexpressed compared to the susceptible accession in the same time period. These results suggest that ACCase1, ACCase3, and CYP81A21 are crucial genes in contributing cyhalofop resistance in this barnyardgrass accession

Target‐site mutations Ile1781Leu and Ile2041Asn in the ACCase2 gene confer resistance to fluazifop‐p‐butyl and pinoxaden herbicides in a johnsongrass accession from Arkansas, USA

Abstract Johnsongrass [Sorghum halepense (L.) Pers.] is a troublesome weed species in different agricultural and non‐agricultural areas. Because of its biology, reproductive system, and seed production, effective management is challenging. An accession with low susceptibility to the acetyl‐CoA carboxylase (ACCase)‐inhibiting herbicides fluazifop‐p‐butyl (fluazifop) and pinoxaden was collected in eastern Arkansas. In this research, the molecular mechanisms responsible for ACCase resistance were investigated. Dose–response experiments showed a resistance factor of 181 and 133 for fluazifop and pinoxaden, respectively. Molecular analysis of both ACCase1 and ACCase2 genes was researched. Nucleotide comparison of ACCase1 between resistant and susceptible accessions showed no single nucleotide polymorphisms. Nonetheless, analysis of ACCase2 in fluazifop‐resistant johnsongrass plants revealed the Ile1781Leu target‐site mutation was dominant (nearly 75%), whereas the majority of pinoxaden‐resistant johnsongrass plants had the Ile2041Asn (60%). Not all sequenced johnsongrass plants displayed a target‐site mutation, suggesting the presence of additional resistance mechanisms. Amplification of ACCase1 and ACCase2 was not responsible for resistance because of the similar values obtained in both resistant and susceptible accessions. Experiments with malathion and NBD‐Cl suggest the presence of herbicide metabolism. Outcomes of this research demonstrated that fluazifop‐ and pinoxaden‐resistant johnsongrass plants displayed a target‐site mutation in ACCase2, but also that non‐target‐site resistance mechanisms would be involved and require a detailed study

Overexpression of <i>Acetyl CoA Carboxylase 1</i> and <i>3</i> (<i>ACCase1</i> and <i>ACCase3</i>), and <i>CYP81A21</i> were related to cyhalofop resistance in a barnyardgrass accession from Arkansas

Barnyardgrass [Echinochloa crus-galli (L.) P. Beauv.] is the most difficult-to-control weed species of rice production systems worldwide. It has evolved resistance to different herbicide sites of action, including the acetyl-CoA carboxylase (ACCase)-inhibiting herbicides. Target-site mutations conferring resistance to ACCase-inhibiting herbicides are well documented; however, the role of the different ACCase genes in conferring resistance to cyhalofop-p-butyl (cyhalofop), an ACCase-inhibiting herbicide, remains poorly understood. This research assessed the contribution of gene amplification and expression of ACCase genes in a cyhalofop-resistant barnyardgrass accession. Additionally, the expression of glutathione-S-transferases (GSTs) and cytochrome P450 monooxygenases (P450s) genes as possible contributors to resistance to cyhalofop were investigated. Results demonstrated that ACCase gene amplification does not contribute to cyhalofop resistance. However, ACCase1 and ACCase3 were found to be overexpressed in the cyhalofop-resistant barnyardgrass accession. At 24 h after cyhalofop treatment, an overexpression of 2.0- and 2.8-fold was detected in ACCase1 and ACCase3, respectively. In addition, CYP81A21 (a P450 gene) was found to be 2.5-fold overexpressed compared to the susceptible accession in the same time period. These results suggest that ACCase1, ACCase3, and CYP81A21 are crucial genes in contributing cyhalofop resistance in this barnyardgrass accession.</p

Screening for detection of wild oat biotypes resistant to fenoxaprop-P-ethyl from Mexico

Avena is one of the world’s most important genera resistant to herbicides. Suspected resistant biotype and susceptible biotype of Avena fatua were tested by screening to confirm the resistance to fenoxaprop-P-ethyl from Mexico. Suspected-resistant population was collected in wheat fields after the herbicide failed to control wild oat and the susceptible one was collected from a field with no herbicide application. Petri dish experiments were performed using different fenoxaprop-P-ethyl concentrations (0, 1, 5, 10 and 40 mM). Experiments were arranged in a completely randomized design with ten replicates. Plumule length in both biotypes decreased as fenoxaprop-P-ethyl concentration increased. However, there was a different response between the R and S biotypes. The EC50 for the resistant biotype was 3.3 mM while for the susceptible biotype was 9.3 mM, showing that the resistant biotype tolerated herbicide concentration approximately three times higher than the susceptible biotype. The results confirm the presence of Avena fatua resistant to fenoxaprop-P-ethyl in Mexico

Target site mutation and reduced translocation are present in a glyphosate-resistant Lolium multiflorum Lam. biotype from Spain

The resistance mechanism of a glyphosate-resistant Lolium multiflorum Lam. biotype collected in Córdoba (Southern Spain) was examined. Resistance Factor values at three different growth stages ranged between 4.77 and 4.91. At 96 hours after treatment (HAT) the S biotype had accumulated seven times more shikimic acid than the R biotype. There were significant differences in translocation of 14C-glyphosate between biotypes, i.e. at 96 HAT, the R biotype accumulated in the treated leaf more than 70% of the absorbed herbicide, in comparison with 59.21% of the S biotype; the R biotype translocated only 14.79% of the absorbed 14C-glyphosate to roots, while in the S population this value was 24.79%. Visualization of 14C-glyphosate by phosphor imaging showed a reduced distribution in the R biotype compared with the S. Glyphosate metabolism was not involved in the resistance mechanism due to both biotypes showing similar values of glyphosate at 96 HAT. Comparison of the EPSPS gene sequences between biotypes indicated that the R biotype has a proline 182 to serine amino acid substitution. In short, the resistance mechanism of the L. multiflorum Lam. biotype is due to an impaired translocation of the herbicide and an altered target site. © 2012 Elsevier Masson SAS.This research was funded by the firm Monsanto and by Spain's MICINN (Project AGL2010-16774). Javier Gil-Humanes acknowledges financial support from the I3P Program from the Consejo Superior de Investigaciones Científicas, which is co-financed by the European Social Fund.Peer Reviewe

Glyphosate efficacy in Avena sterilis and Lolium rigidum populations

Póster presentado en el International workshop on “Glyphosate weed resistance: European status and solutions”, celebrado en Córdoba (España) el 3 y 4 de mayo de 2012.The physical, physiological and molecular basis of tolerance/resistance to glyphosate in Avena sterilis and Lolium rigidum, respectively were examined. Samples of these species were collected from fields with a long history of glyphosate usage (Exposed – “E”) and from areas that were not exposed to herbicides (UnExposed – “UE”). A. sterilis (“E” and “UE”) was found to have roughly 3 times greater natural resistance to glyphosate than L. rigidum (“UE”). “E” population of L. rigidum was found to be 5 times more resistant to glyphosate than the “UE” population. There was no difference in shikimic acid accumulation between the “E” and “UE” population for A. sterilis, but, the “UE” L. rigidum population accumulated 6 times more shikimic acid than the “E”. Both species absorbed 14C-glyphosate differentially, absorption decreased in the following order L. rigidum “UE” > A. sterilis “E” and “UE” > L. rigidum “E”. There were also marked differences between species regarding the amount of 14C-glyphosate translocated from the treated leaf to the rest of plant shoots and roots. Glyphosate translocation from treated leaf to roots was maximal in L. rigidum (“UE”) followed by A. sterilis (“E” and “UE”) and minimal in L. rigidum (“E”). Comparison of the EPSPS gene sequences between L. rigidum populations indicated that the “E” has a serine amino acid substitution at position 182 of the predicted protein instead of the proline amino acid present in the “UE” population.Peer Reviewe

First evidence for a target site mutation in the EPSPS2 gene in glyphosate-resistant Sumatran fleabane from citrus orchards

The glyphosate herbicide has been extensively used for long time periods in woody crops to control a broad range of weeds. The rapid determination of resistant weeds in different woody crops could maintain the efficacy of herbicides and could improve weed management using rotating strategies. Unfortunately Sumatran fleabane has developed a resistance to glyphosate. The mechanism of resistance of Sumatran fleabane is unknown so far. Therefore, here, we studied the resistance of a Sumatran fleabane biotype collected from a citrus orchard, under greenhouse and laboratory conditions. Our results show a resistance factor of 7.4. The resistant biotype absorbed and translocated lower amounts of 14C-glyphosate compared to the susceptible biotype. Moreover, at the molecular level, the target site sequence of the EPSPS2 gene showed a Pro-182-Thr substitution in the resistant biotype. As a consequence, this biotype uses mechanisms of reduced absorption-translocation and target site mutation to resist against glyphosate. This is the first study to report the reduced absorption and a mutation in the EPSPS2 gene in the resistance mechanism in the Conyza genus. © 2013 INRA and Springer-Verlag France.The Monsanto Company and Spain’s MICINN Project (AGL2010-16774) supported this research.Peer Reviewe