First Case of Conyza canadensis from Hungary with Multiple Resistance to Glyphosate and Flazasulfuron

Conyza canadensis is a species invading large areas throughout the world, mainly due to its ability to evolve herbicide resistance. In Hungary, extensive areas have been infested by this species due to the difficulty in controlling it with glyphosate. To determine whether poor control was a result of misapplication or glyphosate resistance, eight suspected glyphosate-resistant C. canadensis populations from different Hungarian regions were studied. In whole-plant dose-response assays with glyphosate, the LD50 and GR50 values (survival and fresh weight reduction at 50% relative to the untreated control, respectively) indicated that resistance was confirmed in five of the eight populations (H-5 population being the most resistant). Additionally, the shikimic acid accumulation tests corroborated the results observed in the dose–response assays. 11 alternative herbicides from six different modes of action (MOA) were applied at field doses as control alternatives on populations H-5 and H-6 (both in the same regions). The H-5 population showed an unexpected resistance to flazasulfuron (ALS-inhibitor). The ALS enzyme activity studies indicated that the I50 for H-5 with flazasulfuron was 63.3 times higher compared to its correspondent susceptible population (H-6). Therefore, the H-5 population exhibited multiple-resistance to flazasulfuron and glyphosate, being the first case reported in Europe for these two MOA

Target-Site and Non-Target Site Resistance (TSR and NTSR) mechanisms in glyphosate-resistant grass weeds

El control de malas hierbas mediante el uso de herbicidas es una de las principales herramientas utilizadas en la agronomía con la finalidad de poder subsistir y alcanzar mayores niveles de producción agrícola. No obstante, el uso repetido de los herbicidas ha ocasionado que múltiples especies hayan evolucionado como resistentes a estos productos. El glifosato es el herbicida con mayores ventas en el mundo, y es utilizado ampliamente en post-emergencia o pre-siembra para el control de malas hierbas dico y monocotiledóneas. El modo de acción de este herbicida es la inhibición de la 5- enolpiruvilshikimato-3-fosfato sintasa (EPSPS), enzima importante en la biosíntesis de aminoácidos esenciales fenilalanina, tirosina y triptófano en las plantas. De acuerdo con “The International Survey of Herbicide Resistant Weeds”, actualmente existen 55 casos de resistencia glifosato reportados en el mundo. Dada la importancia del uso de herbicidas y de un adecuado manejo integrado de malas hierbas, en el presente trabajo se han confirmado en España, Colombia y Brasil, primeros casos de resistencia a glifosato, y se han caracterizado los mecanismos de resistencia para que así se pueda obtener una adecuada decisión en cuanto al control de malas hierbas resistentes. En este trabajo se confirmó el primer caso mundial de resistencia de Bromus rubens, y mediante ensayos de invernadero se detectó que existen alternativas químicas como el propaquizafop y flazasulfuron, dos herbicidas con modo de acción diferente al glifosato. Por otro lado, se caracterizaron por primera vez los mecanismos de resistencia en Echinochloa crus-galli resistente a glifosato en cultivos anuales y perennes de la península ibérica. Se encontró que en esta resistencia está implicada una baja absorción y traslocación del herbicida, además, en una población está implicado el metabolismo de glifosato a metabolitos no tóxicos (ácido amino metil fosfonico (AMPA) y glioxilato). También se encontró que el primer caso de Chloris radiata, en arroces colombianos, era debido a una mutación (Pro- 106-Ser) en el gen que codifica a la enzima EPSPS. Por último, se encontró que una resistencia de Chloris distichophylla en Brasil, era debido a una baja absorción y traslocación del glifosato. Además, mediante estudios con herbicidas alternativos se encontró que productos como el cletodim, quizalofop, diuron, tembotrione o glufositato, pueden ser herramientas útiles para el control de esta gramínea. La caracterización de los mecanismos de resistencia implicados en cada maleza resistente a herbicidas es la mejor herramienta y la base para desarrollar estrategias de manejo integrado de malas hierbas (MIM). El cambio en las estrategias de control de malas hierbas en cultivos españoles, colombianos y brasileños debe incluir herbicidas con modo de acción diferente al glifosato y métodos no químicos para preservar la vida útil del glifosato por más tiempo para el control de malas hierbas en estos países.Weed control using herbicides is one of the main tools used in agronomy in order to persist and achieve higher levels of agricultural production. However, the repeated use of herbicides has caused multiple species to evolve resistance to these products. Glyphosate is the herbicide with the highest sales in the world and is widely used in post-emergence or pre-sowing for the dicot and monocotyledonous control weeds. The mode of action of this herbicide is the inhibition of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), an important enzyme in the biosynthesis of essential amino acids phenylalanine, tyrosine, and tryptophan in plants. According to "The International Survey of Herbicide Resistant Weeds", there are currently 55 cases of glyphosate resistance reported worldwide. Given the importance of herbicide use and proper integrated weed management, the first cases of resistance to glyphosate have been confirmed in Spain, Colombia and Brazil, and the mechanisms of resistance have been characterized to obtain an adequate decision regarding the control of resistant weeds. In this work, the first world case of resistance of Bromus rubens was confirmed, and through greenhouse assays it was detected that there are chemical alternatives such as propaquizafop and flazasulfuron, two herbicides with a different mode of action to glyphosate. On the other hand, the mechanisms of resistance in glyphosate-resistant Echinochloa crus-galli in annual and perennial crops in the Iberian Peninsula were characterized for the first time. It was found that low uptake and translocation of the herbicide is involved in this resistance, and that glyphosate metabolism to non-toxic metabolites (amino methyl phosphonic acid (AMPA) and glyoxylate) is involved in one population. The first case of Chloris radiata in Colombian rice was also found to be due to a mutation (Pro-106-Ser) in the gene encoding the EPSPS enzyme. Finally, a resistance of Chloris distichophylla in Brazil was found to be due to a low uptake and translocation of glyphosate. In addition, through trials with alternative herbicides, it was found that products such as clethodim, quizalofop, diuron, tembotrione or glufositate, can be useful tools for the control of this grassweed. Characterizing resistance mechanisms implied in each herbicide resistant weed is the best tool and the basis to develop integrated weed management (IWM) strategies. The change in weed control strategies in Spanish, Colombian and Brazilian crops should include herbicides with a mode of action different from glyphosate and non-chemical methods to preserve the useful life of glyphosate for a longer time for weed control in these countries

Evidence, Mechanism and Alternative Chemical Seedbank-Level Control of Glyphosate Resistance of a Rigid Ryegrass (Lolium rigidum) Biotype from Southern Spain

Rigid ryegrass (Lolium rigidum) is one of the most troublesome weeds in different crops in the Mediterranean region. A rigid ryegrass biotype from an olive grove in Jaén province (Andalusía, southern Spain), potentially resistant to glyphosate (RG), was tested for its resistance level through dose-response assays using a susceptible biotype (SG). To test the hypothesis of a non-target-site-based resistance, as point mutations are far less common mechanisms of glyphosate resistance, studies were also conducted to elucidate whether resistance was associated with biochemical, metabolism, molecular and/or physiological mechanisms. Alternative herbicide-based control options, including single-herbicide or herbicide mixtures with glyphosate, applied at seedling, tillering or full heading stages, were tested in field experiments for 2 years for their efficacy against rigid ryegrass plants and their effects on the soil seed bank. Resistance levels of the RG biotype were 23- (LD50) and 7-fold (GR50) higher compared to the SG biotype. The SG biotype exhibited a significantly greater shikimic acid accumulation than the RG one. At 96 HAT, 58 and 89% of applied 14C-glyphosate was up taken by leaves of RG and SG biotype plants, respectively, and, at this time, a significantly higher proportion of the glyphosate taken up by the treated leaf remained in its tissue in RG plants compared to the SG ones. The RG biotype did not reveal any point mutation in the glyphosate target site EPSP synthase. Overall, results confirmed reduced glyphosate uptake and translocation as being the mechanism involved in glyphosate resistance in the RG biotype. RG biotype responses to the alternative treatments tested in situ indicated that herbicide applications at the later growth stage tended to be less effective in terms of immediate effects on population size than earlier applications, and that only in some cases, the removal of at least 85% of the RG biotype was achieved. However, with few exceptions, the alternative treatments tested appeared to be highly effective in reducing the seed bank irrespective of the growth stage. The frequency of the resistant phenotype in the progeny of surviving plants of the RG biotype was dependent on treatment. Results suggest that a potential exists for effective management of glyphosate-resistant rigid ryegrass in olive groves in southern Spain.This work was funded by the Spanish Ministry of Economy and Competitiveness (AGL2016-78944-R), and partially by Monsanto Europe S.A. (Brussels)

Distribution of Glyphosate-Resistance in Echinochloa crus-galli Across Agriculture Areas in the Iberian Peninsula

The levels of resistance to glyphosate of 13 barnyard grass (Echinochloa crus-galli) populations harvested across different agriculture areas in the Southern Iberian Peninsula were determined in greenhouse and laboratory experiments. Shikimate accumulation fast screening separated the populations regarding resistance to glyphosate: susceptible (S) E2, E3, E4, and E6 and resistant (R) E1, E5, E7, E8, E9, E10, E11, E12, and E13. However, resistance factor (GR50 E1–E13/GR50 E6) values separated these populations into three groups: (S) E2, E3, E4, and E6, (R) E1, E5, E7, E8, and E9, and very resistant (VR) E10, E11, E12, and E13. 14C-glyphosate assays performed on two S populations (E2 and E6) showed greater absorption and translocation than those found for R (E7 and E9) and VR (E10 and E12) populations. No previous population metabolized glyphosate to amino methyl phosphonic acid (AMPA) and glyoxylate, except for the E10 population that metabolized 51% to non-toxic products. The VR populations showed two times more 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) activity without herbicide than the rest, while the inhibition of the EPSPS activity by 50% (I50) required much higher glyphosate in R and VR populations than in S populations. These results indicated that different target-site and non-target-site resistance mechanisms were implicated in the resistance to glyphosate in E. crus-galli. Our results conclude that resistance is independent of climate, type of crop, and geographic region and that the level of glyphosate resistance was mainly due to the selection pressure made by the herbicide on the different populations of E. crus-galli studied

Cross- and multiple-resistance of weeds to herbicides

La agricultura es una de las principales potencias económicas, produciendo desarrollo y riqueza a nivel mundial. Este sector está afectado por diversos factores, tanto bióticos como abióticos, los cuales se traducen en elevadas pérdidas de rendimiento y costes en los cultivos. Los principales factores bióticos que afectan a la agricultura son las enfermedades, plagas y malas hierbas. Las malas hierbas son plantas que crecen de forma predominante en situaciones alteradas por el hombre, y que no resultan deseables para él en un lugar y momento determinado. Los herbicidas representan en la actualidad un papel imprescindible en el control de malas hierbas, siendo la herramienta más efectiva que se haya desarrollado, controlando alrededor del 99% de malas hierbas. Desafortunadamente, la era dorada de los herbicidas como única herramienta de control se ha visto truncada a causa de la aparición de malas hierbas resistentes a herbicidas por el uso abusivo de estos. El aumento exponencial de malas hierbas con resistencia se produce en una situación en la que durante los últimos 25 años no se han desarrollado nuevos modos de acción. La resistencia a herbicidas es el resultado de la adaptación evolutiva de las malas hierbas a las sucesivas aplicaciones herbicidas, y es sin duda, una de las principales preocupaciones en la agricultura moderna. Desafortunadamente, las malas hierbas pueden desarrollar no solo resistencia a un herbicida, sino que pueden desarrollar resistencia cruzada y/o múltiple. La comunidad centrada en la malherbologia presenta la preocupación de que los agricultores podrían enfrentarse a la perdida de los herbicidas como herramientas eficaces y económicas en las que se basa la agricultura productiva moderna. Este preocupante escenario comienza con la dependencia exclusiva del uso de un solo herbicida, ya sea por ejemplo glifosato en cultivos perennes, o diclofop en cultivos anuales. Ante esta situación, los agricultores cambian de herbicida, pero en muchas ocasiones no cambian de modo de acción, por lo que el problema no es solventado. En otras ocasiones, se cambia de modo de acción pero no es alternado con otros herbicidas en las sucesivas aplicaciones, apareciendo nuevamente la resistencia. Debido a la repercusión que ha ocasionado la resistencia de malas hierbas a herbicidas, así como la aparición de resistencia cruzada y/o múltiple, en este trabajo se han determinado los mecanismos de resistencia involucrados en poblaciones de Lolium rigidum resistente a glifosato en Francia [Capítulo II] y España, así como ofrecer alternativas químicas para obtener un control optimo, y poder controlar la resistencia [Capítulo III]. Además, se caracterizó la tolerancia natural de Avena sterilis a glifosato en el sur de España [Capítulo IV], sirviendo de ejemplo de que algunas malas hierbas no son controladas por glifosato, no por haber desarrollado resistencia sino por ser tolerantes. En el capítulo V se determinó la múltiple resistencia a herbicidas no selectivos en especies del genero Lolium spp. en la Península Ibérica, a causa de no alternar diferentes modos de acción en las sucesivas aplicaciones. Del mismo modo, en el Capítulo VI se caracterizó la resistencia cruzada a herbicidas pertenecientes a los inhibidores de la ACCasa (acetil- CoA carboxilasa) en Cynosurus echinatus procedente de Chile, a causa de utilizar herbicidas de diferentes familias químicas, pero todas ellas pertenecientes al mismo modo de acción. Se determinó el primer caso de resistencia francés en L. rigidum en el que se conocen los mecanismos implicados. Los resultados mostraron que la resistencia a glifosato en la población resistente francesa de L. rigidum se debe en parte a la reducción de absorción y translocación de glifosato en relación con la población susceptible, así como a una mutación en el gen que codifica la EPSPS. Por el contrario, la resistencia a glifosato en la población resistente española de L. rigidum se debe en parte a la reducción de absorción y translocación de glifosato, y no se encontró ninguna mutación en el gen que codifica la EPSPS. Los ensayos de campo determinaron que es posible obtener un control eficaz de L. rigidum utilizando diferentes modos de acción, así como la reducción del banco de semillas resistentes a glifosato. Las prospecciones realizadas mostró resultados homogéneos entre todas las accesiones de A. sterilis recogidas y, por lo tanto, todas ellas tienen el mismo nivel de tolerancia innata al glifosato, siendo los mecanismos fuera del sitio de acción los implicados en la tolerancia innata a glifosato en A. sterilis. Esto es probablemente debido en parte a una menor absorción / translocación del herbicida y metabolismo de glifosato. Estudios moleculares confirmaron que tres especies de malas hierbas de Lolium resistentes a glifosato (L. rigidum, L. perenne, y L. multiflorum) recogidas de cultivos perennes en la Península Ibérica también han desarrollado resistencia múltiple a los herbicidas glufosinato y oxifluorfen. Este estudio identificó el primer caso de resistencia a oxifluorfen en una gramínea. Los ensayos in vitro y de dosisrespuesta de ACCasa determinaron la resistencia cruzada a los herbicidas de las familias químicas APP, CHD y PPZ en C. echinatus. Los estudios de secuenciación de ADN confirmaron que la resistencia cruzada de C. echinatus a los inhibidores de ACCasa ha sido conferida por las mutaciones puntuales Ile-2041-Asn y Cys-2088-Arg.Agriculture is one of the main economic powers, producing development and wealth worldwide. This sector is affected by several factors, both biotic and abiotic, which have resulted in high yield and crop cost losses. The main biotic factors affecting agriculture are diseases, pests, and weeds. Weeds are plants that predominantly grow in situations altered by man, and are not desirable for at any time and given place. To date, herbicides play an essential role in weed control, being the most effective tool developed, controlling about 99% of weeds. Unfortunately, the golden era of herbicides as the only tool has been cut short by the herbicide-resistant weeds due to abusive use. The exponential increase of resistant weeds occurs in a situation where no new action modes have been developed during the last 25 years. Herbicide resistance is the result of the evolutionary adaption of weeds to successive herbicide applications, and it is undoubtedly one of the main concerns in modern agriculture. Unfortunately, weeds can develop not only resistance to one herbicide, but also they can develop cross- and multiple-resistance. The weed research community is concerned that farmers may be faced with the herbicide loss as an effective and economic tool on which modern productive agriculture is based. This worrying scenario begins with the exclusive use of a single herbicide, whether for example glyphosate in perennial crops, or diclofop in annual crops. In this situation, farmers change to another herbicide, but in many cases, they do not change the action mode, so the problem is not solved. At other times, they change the action mode, but it is not alternated with other action modes in the successive applications, exhibiting resistance again. Due to the herbicide-resistant weed impact, as well as the occurrence of cross- and multiple-resistance, this work has determined the mechanisms of resistance involved in glyphosate-resistant populations of Lolium rigidum from France [Chapter II], and Spain, as well as to offer chemical alternatives to control the resistance [Chapter III]. Also, the natural tolerance of Avena sterilis to glyphosate in southern Spain has been characterized [Chapter IV], offering as an example that some weeds are not controlled by glyphosate, not because of developed resistance, but because they are tolerant. In Chapter V, the multiple-resistance to non-selective herbicides in species of Lolium spp. genus from the Iberian Peninsula, due to not alternating different action modes in the successive applications has been reported. In the same way, cross-resistance to herbicides belonging to the ACCase (acetyl-CoA carboxylase)-inhibitors in Cynosurus echinatus from Chile has been characterized in Chapter VI, because of the use of herbicides of different chemical families, but all of them belonging to the same action mode. The first case of French resistance of L. rigidum has been determined in which the mechanisms involved are known. The results showed that glyphosate resistance in the French resistant population of L. rigidum was due in part to the reduction of glyphosate absorption and translocation in relation to the susceptible one, as well as to a mutation in the gene encoding EPSPS. In contrast, glyphosate resistance in the Spanish population of L. rigidum was due to reduced absorption and translocation, and no mutation was found in the gene encoding EPSPS. Field trials determined that it is possible to obtain an effective control of L. rigidum using different action modes, as well as the reduction of the glyphosate resistant seed bank. The surveys carried out showed homogenous results among all of the A. sterilis accessions collected; therefore, all of them have the same level of innate tolerance of glyphosate, being the non-target-site resistance the mechanism involved. This was due in part to a lower absorption and translocation, and glyphosate metabolism. Molecular studies confirmed that three species of glyphosate-resistant Lolium (L. rigidum, L. multiflorum, and L. perenne) collected from perennial crops have also developed multiple-resistance to glufosinate and oxyfluorfen. This study reported the first case of resistance to oxyfluorfen in grass. The in vivo and dose-response assays of ACCase determined the cross-resistance to the herbicides of the APP, CHD, and PPZ chemical families in C. echinatus. DNA sequencing studies confirmed that crossresistance to ACCase-inhibitors in C. echinatus has been conferred by Ile-2041-Asn and Cys-2088-Arg point mutations

Target site as the main mechanism of resistance to imazamox in a Euphorbia heterophylla biotype

Euphorbia heterophylla is a weed species that invades extensive crop areas in subtropical regions of Brazil. This species was previously controlled by imazamox, but the continuous use of this herbicide has selected for resistant biotypes. Two biotypes of E. heterophylla from southern Brazil, one resistant (R) and one susceptible (S) to imazamox, were compared. The resistance of the R biotype was confrmed by dose-response assays since it required 1250.2g ai ha−1 to reduce the fresh weight by 50% versus 7.4g ai ha−1 for the S biotype. The acetolactate synthase (ALS) enzyme activity was studied using ALS-inhibiting herbicides from fve diferent chemical families. The R biotype required the highest concentrations to reduce this enzyme activity by 50%. A Ser653Asn mutation was found in the ALS gene of the R biotype. The experiments carried out showed that imazamox absorption and metabolism were not involved in resistance. However, greater 14C-imazamox root exudation was found in the R biotype (~70% of the total absorbed imazamox). Target site mutation in the ALS gene is the principal mechanism that explains the imazamox resistance of the R biotype, but root exudation seems to also contribute to the resistance of this biotyp

Non-Target Site Mechanisms Endow Resistance to Glyphosate in Saltmarsh Aster (Aster squamatus)

Of the six-glyphosate resistant weed species reported in Mexico, five were found in citrus groves. Here, the glyphosate susceptibility level and resistance mechanisms were evaluated in saltmarsh aster (Aster squamatus), a weed that also occurs in Mexican citrus groves. The R population accumulated 4.5-fold less shikimic acid than S population. S plants hardly survived at 125 g ae ha−1 while most of the R plants that were treated with 1000 g ae ha−1, which suffered a strong growth arrest, showed a vigorous regrowth from the third week after treatment. Further, 5-enolpyruvylshikimate-3-phosphate basal and enzymatic activities did not diverge between populations, suggesting the absence of target-site resistance mechanisms. At 96 h after treatment, R plants absorbed ~18% less glyphosate and maintained 63% of the 14C-glyphsoate absorbed in the treated leaf in comparison to S plants. R plants metabolized twice as much (72%) glyphosate to amino methyl phosphonic acid and glyoxylate as the S plants. Three non-target mechanisms, reduced absorption and translocation and increased metabolism, confer glyphosate resistance saltmarsh aster. This is the first case of glyphosate resistance recorded for A. squamatus in the world

Cross- and multiple-resistance of weeds to herbicides

La agricultura es una de las principales potencias económicas, produciendo desarrollo y riqueza a nivel mundial. Este sector está afectado por diversos factores, tanto bióticos como abióticos, los cuales se traducen en elevadas pérdidas de rendimiento y costes en los cultivos. Los principales factores bióticos que afectan a la agricultura son las enfermedades, plagas y malas hierbas. Las malas hierbas son plantas que crecen de forma predominante en situaciones alteradas por el hombre, y que no resultan deseables para él en un lugar y momento determinado. Los herbicidas representan en la actualidad un papel imprescindible en el control de malas hierbas, siendo la herramienta más efectiva que se haya desarrollado, controlando alrededor del 99% de malas hierbas. Desafortunadamente, la era dorada de los herbicidas como única herramienta de control se ha visto truncada a causa de la aparición de malas hierbas resistentes a herbicidas por el uso abusivo de estos. El aumento exponencial de malas hierbas con resistencia se produce en una situación en la que durante los últimos 25 años no se han desarrollado nuevos modos de acción. La resistencia a herbicidas es el resultado de la adaptación evolutiva de las malas hierbas a las sucesivas aplicaciones herbicidas, y es sin duda, una de las principales preocupaciones en la agricultura moderna. Desafortunadamente, las malas hierbas pueden desarrollar no solo resistencia a un herbicida, sino que pueden desarrollar resistencia cruzada y/o múltiple. La comunidad centrada en la malherbologia presenta la preocupación de que los agricultores podrían enfrentarse a la perdida de los herbicidas como herramientas eficaces y económicas en las que se basa la agricultura productiva moderna. Este preocupante escenario comienza con la dependencia exclusiva del uso de un solo herbicida, ya sea por ejemplo glifosato en cultivos perennes, o diclofop en cultivos anuales. Ante esta situación, los agricultores cambian de herbicida, pero en muchas ocasiones no cambian de modo de acción, por lo que el problema no es solventado. En otras ocasiones, se cambia de modo de acción pero no es alternado con otros herbicidas en las sucesivas aplicaciones, apareciendo nuevamente la resistencia. Debido a la repercusión que ha ocasionado la resistencia de malas hierbas a herbicidas, así como la aparición de resistencia cruzada y/o múltiple, en este trabajo se han determinado los mecanismos de resistencia involucrados en poblaciones de Lolium rigidum resistente a glifosato en Francia [Capítulo II] y España, así como ofrecer alternativas químicas para obtener un control optimo, y poder controlar la resistencia [Capítulo III]. Además, se caracterizó la tolerancia natural de Avena sterilis a glifosato en el sur de España [Capítulo IV], sirviendo de ejemplo de que algunas malas hierbas no son controladas por glifosato, no por haber desarrollado resistencia sino por ser tolerantes. En el capítulo V se determinó la múltiple resistencia a herbicidas no selectivos en especies del genero Lolium spp. en la Península Ibérica, a causa de no alternar diferentes modos de acción en las sucesivas aplicaciones. Del mismo modo, en el Capítulo VI se caracterizó la resistencia cruzada a herbicidas pertenecientes a los inhibidores de la ACCasa (acetil- CoA carboxilasa) en Cynosurus echinatus procedente de Chile, a causa de utilizar herbicidas de diferentes familias químicas, pero todas ellas pertenecientes al mismo modo de acción. Se determinó el primer caso de resistencia francés en L. rigidum en el que se conocen los mecanismos implicados. Los resultados mostraron que la resistencia a glifosato en la población resistente francesa de L. rigidum se debe en parte a la reducción de absorción y translocación de glifosato en relación con la población susceptible, así como a una mutación en el gen que codifica la EPSPS. Por el contrario, la resistencia a glifosato en la población resistente española de L. rigidum se debe en parte a la reducción de absorción y translocación de glifosato, y no se encontró ninguna mutación en el gen que codifica la EPSPS. Los ensayos de campo determinaron que es posible obtener un control eficaz de L. rigidum utilizando diferentes modos de acción, así como la reducción del banco de semillas resistentes a glifosato. Las prospecciones realizadas mostró resultados homogéneos entre todas las accesiones de A. sterilis recogidas y, por lo tanto, todas ellas tienen el mismo nivel de tolerancia innata al glifosato, siendo los mecanismos fuera del sitio de acción los implicados en la tolerancia innata a glifosato en A. sterilis. Esto es probablemente debido en parte a una menor absorción / translocación del herbicida y metabolismo de glifosato. Estudios moleculares confirmaron que tres especies de malas hierbas de Lolium resistentes a glifosato (L. rigidum, L. perenne, y L. multiflorum) recogidas de cultivos perennes en la Península Ibérica también han desarrollado resistencia múltiple a los herbicidas glufosinato y oxifluorfen. Este estudio identificó el primer caso de resistencia a oxifluorfen en una gramínea. Los ensayos in vitro y de dosisrespuesta de ACCasa determinaron la resistencia cruzada a los herbicidas de las familias químicas APP, CHD y PPZ en C. echinatus. Los estudios de secuenciación de ADN confirmaron que la resistencia cruzada de C. echinatus a los inhibidores de ACCasa ha sido conferida por las mutaciones puntuales Ile-2041-Asn y Cys-2088-Arg.Agriculture is one of the main economic powers, producing development and wealth worldwide. This sector is affected by several factors, both biotic and abiotic, which have resulted in high yield and crop cost losses. The main biotic factors affecting agriculture are diseases, pests, and weeds. Weeds are plants that predominantly grow in situations altered by man, and are not desirable for at any time and given place. To date, herbicides play an essential role in weed control, being the most effective tool developed, controlling about 99% of weeds. Unfortunately, the golden era of herbicides as the only tool has been cut short by the herbicide-resistant weeds due to abusive use. The exponential increase of resistant weeds occurs in a situation where no new action modes have been developed during the last 25 years. Herbicide resistance is the result of the evolutionary adaption of weeds to successive herbicide applications, and it is undoubtedly one of the main concerns in modern agriculture. Unfortunately, weeds can develop not only resistance to one herbicide, but also they can develop cross- and multiple-resistance. The weed research community is concerned that farmers may be faced with the herbicide loss as an effective and economic tool on which modern productive agriculture is based. This worrying scenario begins with the exclusive use of a single herbicide, whether for example glyphosate in perennial crops, or diclofop in annual crops. In this situation, farmers change to another herbicide, but in many cases, they do not change the action mode, so the problem is not solved. At other times, they change the action mode, but it is not alternated with other action modes in the successive applications, exhibiting resistance again. Due to the herbicide-resistant weed impact, as well as the occurrence of cross- and multiple-resistance, this work has determined the mechanisms of resistance involved in glyphosate-resistant populations of Lolium rigidum from France [Chapter II], and Spain, as well as to offer chemical alternatives to control the resistance [Chapter III]. Also, the natural tolerance of Avena sterilis to glyphosate in southern Spain has been characterized [Chapter IV], offering as an example that some weeds are not controlled by glyphosate, not because of developed resistance, but because they are tolerant. In Chapter V, the multiple-resistance to non-selective herbicides in species of Lolium spp. genus from the Iberian Peninsula, due to not alternating different action modes in the successive applications has been reported. In the same way, cross-resistance to herbicides belonging to the ACCase (acetyl-CoA carboxylase)-inhibitors in Cynosurus echinatus from Chile has been characterized in Chapter VI, because of the use of herbicides of different chemical families, but all of them belonging to the same action mode. The first case of French resistance of L. rigidum has been determined in which the mechanisms involved are known. The results showed that glyphosate resistance in the French resistant population of L. rigidum was due in part to the reduction of glyphosate absorption and translocation in relation to the susceptible one, as well as to a mutation in the gene encoding EPSPS. In contrast, glyphosate resistance in the Spanish population of L. rigidum was due to reduced absorption and translocation, and no mutation was found in the gene encoding EPSPS. Field trials determined that it is possible to obtain an effective control of L. rigidum using different action modes, as well as the reduction of the glyphosate resistant seed bank. The surveys carried out showed homogenous results among all of the A. sterilis accessions collected; therefore, all of them have the same level of innate tolerance of glyphosate, being the non-target-site resistance the mechanism involved. This was due in part to a lower absorption and translocation, and glyphosate metabolism. Molecular studies confirmed that three species of glyphosate-resistant Lolium (L. rigidum, L. multiflorum, and L. perenne) collected from perennial crops have also developed multiple-resistance to glufosinate and oxyfluorfen. This study reported the first case of resistance to oxyfluorfen in grass. The in vivo and dose-response assays of ACCase determined the cross-resistance to the herbicides of the APP, CHD, and PPZ chemical families in C. echinatus. DNA sequencing studies confirmed that crossresistance to ACCase-inhibitors in C. echinatus has been conferred by Ile-2041-Asn and Cys-2088-Arg point mutations

Resistance to auxin mimics and EPSPS and ALS inhibitor herbicides in dicotyledonous weeds. Resistance mechanisms

Actualmente el control químico de las malas hierbas es la principal herramienta utilizada en el mundo dentro y fuera de los cultivos. Desde la introducción de los herbicidas se ha sometido a una gran cantidad de malas hierbas a una selección constate que ha dado como resultado la rápida evolución de la resistencia a los herbicidas. La ciencia de las malas hierbas ha establecido y sigue desarrollando protocolos para conocer y dilucidar los mecanismos que confieren resistencia a uno o varios herbicidas y especies de malas hierbas. La resistencia a herbicidas se divide en: resistencia dentro del sitio de acción (TSR) y resistencia fuera del sitio de acción (NTSR). La resistencia dentro del sitio de acción se atribuye generalmente a mutaciones en el gen que codifica la enzima objetivo del herbicida que provoca una disminución de su afinidad con el herbicida. La resistencia fuera del sitio de acción es el producto de la evolución más avanzada de las malas hierbas ya este impide que el herbicida llegue adecuadamente a su sitio de acción. Dentro de este grupo encontramos la absorción y translocación reducida, el secuestro vacuolar y el metabolismo de los herbicidas. Las malas hierbas pueden tener un solo mecanismo de resistencia o acumular varios mecanismos que se traduce en resistencia múltiple a varios herbicidas y que representan un gran desafío para la sostenibilidad de los herbicidas en la agricultura. En este trabajo de investigación se da a conocer el primer caso de resistencia a glifosato en Amaranthus palmeri que involucra exclusivamente mecanismos NTSR como la absorción y translocación diferenciada entre poblaciones. También se encontró resistencia a 2,4-D en seis especies dicotiledóneas donde el metabolismo mejorado y la translocación reducida son los responsables de la resistencia a este imitador de auxinas. Por otro lado, se estudió una población de Conyza bonariensis, donde por primera vez en el mundo se informa de la aparición de resistencia múltiple a herbicidas inhibidores de la aceto lactato sintasa (ALS) y la 5-enolpiruvylshikimato-3-fosfato sintasa (EPSPS), desviadores de electrones del fotosistema I (PSI), inhibidores del fotosistema II (PSII) y herbicidas imitadores de auxinas. Conocer y estudiar los mecanismos de resistencia a fondo es imprescindible para comprender la resistencia y construir soluciones que permitan desarrollar sistemas agrícolas sostenibles. El manejo integrado de malas hierbas (IWM) debe jugar un papel importante para disminuir esa presión de selección al que las malas hierban se sometido.Chemical control of weeds is currently the main tool used in the world inside and outside of crops. Since the introduction of herbicides, large number of weeds have been subjected to constant selection pressureresulting to the rapid evolution of herbicide resistance. Weed science has established and continues to develop protocols to test and know the mechanisms that confer resistance for various herbicides and weed species. Herbicide resistance is classified into target site resistance (TSR) and non-target site resistance (NTSR). Target site resistance is generally attributed to mutations in the gene encoding the herbicide target enzyme that cause a decrease in its affinity for the herbicide. Non target site resistance is the product of the more advanced evolution and prevents the herbicide from reaching its site of action correctly. Within this group are reduced absorption and translocation, vacuolar sequestration and metabolism of herbicides. Weeds have a single resistance mechanism or accumulate several mechanisms that result in multiple resistance to several herbicides and that represent a major challenge to the sustainability of herbicides in agriculture. In this research work we report the first case of glyphosate resistance in Amaranthus palmeri involving exclusively NTSR mechanisms such as differential absorption and translocation between populations. Resistance to 2,4-D was also found in six dicot species in which enhanced metabolism and reduced translocation are responsible for resistance to this auxin mimic. On the other hand, a population of Conyza bonariensis was studied, where for the first time in the world multiple resistance to acetolactate synthase (ALS) and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitor herbicides, photosystem I electron diverters, photosystem II inhibitors and auxin mimic herbicides. Knowledge and study of resistance mechanisms is essential to understand resistance and build solutions for the development of sustainable agricultural systems. Integrated weed management (IWM) has an important role to play in reducing the selection pressure to which weeds have been subjected