From greenhouse to field practice : herbicide resistance detection using chlorophyll-fluorescence-imaging technology

All over the world, herbicide resistance has developed to one of the most important barriers in weed control, making the implementation of the weed control strategy more complicated. There is an intense need for a rapid, cheap and reliable method to conduct in field detection of herbicide resistant weed populations. In the current thesis with the use of chlorophyll fluorescence imaging technology, such a method is implemented and tested in field conditions. A series of experiments were designed and carried out. The data gathered from these experiments were compiled under three paper articles. Paper 1. A greenhouse experiment was conducted to verify if the parameter, Maximal Photosystem II Quantum Yield (Fv/Fm), could possibly indicate the herbicide efficacy. The chlorophyll-fluorescence-imaging sensor, Weed PAM®, was selected for the measurements. In the first part it was investigated if the Fv/Fm value could differentiate between herbicide sensitive and resistant plants. In the second part two important abiotic stress factors were tested if they affected the Fv/Fm value. I) Six herbicides were tested on herbicide sensitive and resistant Alopecurus myosuroides populations; II) Water shortage and nitrogen deficiency were applied on a herbicide sensitive population to observe their influence on the plants. The sensitive plants presented significantly lower Fv/Fm values than the resistant plants 3 days after treatment (DAT) for the ALS and ACCase inhibitors. On the same day, and for the same treatments the Fv/Fm values of the resistant plants were not affected and similar to the control. Appling a PS II inhibitor reduced the Fv/Fm values of both sensitive and resistant plants rapidly. Yet, sensitive and resistant plants could clearly be separated on 4 DAT based on the different Fv/Fm values. On the other hand, nitrogen deficiency did not influence the photosystem II measurements. Water shortage reduced rapidly the Fv/Fm value of the plants seven days after the application, yet at this point plant symptoms included the death of the plants. According to this experiment, the Weed PAM® sensor has proved its capability to identify the sensitive and resistant A. myosuroides populations shortly after the herbicide application. Paper 2. A verification of the above results was made under field conditions for different A. myosuroides populations and different locations. On the first part 50 populations in total including both sensitive and herbicide resistant populations were tested. The second part field experiments were conducted in ten locations around Germany over two years with the local field population mix. It was investigated if the Weed PAM® sensor could separate between herbicide sensitive and resistant A. myosuroides populations 5 DAT. The different populations were sown in a winter wheat field. Two ACCase- and three ALS- inhibitors were applied. In all herbicide treatments, Fv/Fm values of A. myosuroides were significantly lower than the untreated plants at the 5 DAT. For each location, measurements were conducted at 5 DAT. A visual measurement, to verify the result, was carried out at 21 DAT. In both cases, 95% of the plants were correctly identified as sensitive or resistant. This demonstrated the ability of the Weed PAM® sensor to conduct in field real time detection of herbicide resistant A. myosuroides populations shortly after treatment. Paper 3. Greenhouse and field experiments were carried out to investigate if the chlorophyll fluorescence of soybean plants was altered, under herbicide stress. Herbicide combinations including inhibitors of PS II, DOXP synthase, cell division and microtubule assembly were selected for different pre-emergence treatments. Herbicide combinations including inhibitors of PS II, ALS and ACCase were applied in post-emergence treatments. Chlorophyll fluorescence was measured from the emergence of soybeans until the three/four-leaf stage. Furthermore the stress effect of the different treatments on the soybean plants was determined by measuring their dry biomass. In the greenhouse, post-emergence treatments with ALS and ACCase inhibitors did not seem to induce stress on the soybean plants. As expected, it originally demonstrated low Fv/Fm values when stressed by PS II inhibitors. But the PS II system recovered soon, one week after emergence. Stress induced by other pre-emergence herbicides occurred one week after emergence and lasted longer than the stress induced by the PS II inhibitors. Dry biomass collaborated with the sensor result. Based on the current thesis, the Weed PAM® system can be an important tool in the identification of herbicide resistant weed populations, in a timely manner. It has proven its capabilities both in A. myosuroides as a weed and in soybean plants. This technology will help farmers to take more suitable weed control strategies, as well as less economic and environmental risks.Die weltweite Zunahme an Herbizidresistenzen stellt eine der größten Herausforderungen der heutigen Unkrautbekämpfung dar. Die heutige Landwirtschaft verlangt eine effiziente, kostengünstige und zuverlässige Methode um Herbizidresistenzen an Unkräutern direkt im Feld zu erkennen. Hierzu wurden mehrere Studien im Gewächshaus und unter Feldbedingungen durchgeführt, die in drei Artikeln veröffentlicht wurden. Experiment 1. In diesem Teil der Arbeit wurde ein Gewächshausexperiment mit dem bildgebenden Chlorophyllfluoreszenz-Sensor Weed PAM® durchgeführt. Um die Effektivität von Herbiziden festzustellen wurde der vielversprechende Parameter Maximaler Photosystem II Quantenertrag (Fv/Fm) mit dem Sensor gemessen. Im ersten Versuch wurden sechs verschiedene Herbizide an sensitiven sowie resistenten Alopecurus myosuroides Populationen getestet. Im zweiten Versuch wurden sensitive Populationen Wasserknappheit und Stickstoffmangel ausgesetzt, um deren Stressreaktionen zu beobachten. Dieser Versuch trug dazu bei die Einflüsse von abiotischen Faktoren auf die Fv/Fm-Werte zu erkennen. Die Ergebnisse zeigten, dass 3 Tage nach einer Behandlung (TNB) mit ALS- und ACCase-Hemmern die sensitiven Pflanzen signifikant geringere Fv/Fm-Werte aufwiesen, als die resistenten Populationen. Es zeigte sich nur ein geringer Einfluss des Herbizidstresses auf das Photosystem II der resistenten Pflanzen nach der Behandlung mit ALS- und ACCase-Hemmern. Die Fv/Fm-Werte sensitiver und resistenter Pflanzen fielen unter dem Einfluss von PS II Hemmern jedoch rapide ab. 4 TNB zeigte sich, dass die Fv/Fm-Werte der beiden Populationen sich signifikant unterschieden. Stickstoffmangel hatte während der Messungen keinen signifikanten Einfluss auf das Photosystem II, wohingegen sieben Tage nach Initiierung der Wasserknappheit eine schnelle Reduktion der Fv/Fm-Werte aufgetreten ist. Nach den Ergebnissen dieses Experimentes ist der Weed PAM® Sensor dazu in der Lage kurz nach einer Herbizidbehandlung sensitive und resistente Populationen von A. myosuroides zu erkennen. Experiment 2. Dieses Experiment untersuchte die Erkennung von Herbizideffektivität auf sensitive Pflanzen unter Feldbedingungen mithilfe des Weed PAM® Sensors. Zudem wurde getestet, ob der Sensor in der Lage ist unter diesen Bedingungen auch herbizidresistente A.myosuroides Populationen 5 TNB festzustellen. Auf einem Winterweizenschlag wurde eine herbizidsensitive Population von A. myosuroides ausgesät und mit zwei ACCase- und drei ALS-Hemmern behandelt. Die Fv/Fm-Werte der A.myosuroides Pflanzen waren 5 TNB in allen Herbizidbehandlungen signifikant geringer im Vergleich zu den unbehandelten Pflanzen. Innerhalb von 2 Jahren wurden in einem weiteren Experiment insgesamt 50 sensitive und resistente Populationen an zehn Standorten getestet. Eine visuelle Bonitur erfolgte 21 TNB. Die Ergebnisse zeigten, dass 95% der Erkennungen korrekt durchgeführt wurden. Dies zeigt die hohe Genauigkeit des Weed PAM® Sensors für eine direkte Herbizidresistenz-Erkennung von A.myosuroides Populationen kurz nach der Applikation unter Feldbedingungen. Experiment 3. In diesem Teil der Arbeit wurden in Gewächshaus- und Feldversuchen Sojabohnen einem Herbizidstress ausgesetzt, um zu untersuchen, ob sich die Chlorophyllfluoreszenz-Emissionen nach einer Herbizidapplikation ändern. Herbizid-Kombinationen mit Hemmern des PS II-Systems, der DOXP Synthase und der Zellteilung, sowie des Mikrotubuliaufbaus stellten die Vorauflauf-Varianten dar. Die Nachauflauf-Varianten bestanden aus Herbizidmischungen mit PS II, ALS- und ACCase hemmern. Die Chlorophyllfluoreszenz wurde direkt nach dem Auflaufen der Sojabohnen bis zum 3-4 Blatt Stadium gemessen. Durch die Messung der Trockenmasse der Sojabohnen wurde das Stressniveau bestimmt. Im Gewächshausexperiment wurde beobachtet, dass kein Stress durch die Nachauflauf-Varianten in den Sojabohnen induziert wurde. Pflanzen, die durch PS II-Hemmer gestresst wurden, zeigten nach dem Auflaufen geringe Fv/Fm-Werte. Das Photosystem II der Pflanzen erholte sich jedoch innerhalb einer Woche auf das Niveau der unbehandelten Kontrolle. Der Stress durch andere Nachauflauf-Varianten trat eine Woche nach dem Auflauf auf und dauerte länger an als die Variante mit PS II-Hemmern. Die Aufnahmen der Trockenmasse bestätigten die Erkenntnisse der auf Chlorophyllfluoreszenz basierten Stresserkennung. Unter Zuhilfenahme des Weed PAM® Systems ist es Landwirten möglich die richtigen Unkrautbekämpfungsmaßnahmen noch in derselben Vegetationsperiode zu ergreifen in der das Resistenzproblem in ihrem Feld erkannt wurde. Zusammenfassend hilft diese neue Technologie den Landwirten geeignetere Unkrautbekämpfungsstrategien zu entwickeln und geringere ökonomische und ökologische Risiken einzugehen

From greenhouse to field practice : herbicide resistance detection using chlorophyll-fluorescence-imaging technology

All over the world, herbicide resistance has developed to one of the most important barriers in weed control, making the implementation of the weed control strategy more complicated. There is an intense need for a rapid, cheap and reliable method to conduct in field detection of herbicide resistant weed populations. In the current thesis with the use of chlorophyll fluorescence imaging technology, such a method is implemented and tested in field conditions. A series of experiments were designed and carried out. The data gathered from these experiments were compiled under three paper articles. Paper 1. A greenhouse experiment was conducted to verify if the parameter, Maximal Photosystem II Quantum Yield (Fv/Fm), could possibly indicate the herbicide efficacy. The chlorophyll-fluorescence-imaging sensor, Weed PAM®, was selected for the measurements. In the first part it was investigated if the Fv/Fm value could differentiate between herbicide sensitive and resistant plants. In the second part two important abiotic stress factors were tested if they affected the Fv/Fm value. I) Six herbicides were tested on herbicide sensitive and resistant Alopecurus myosuroides populations; II) Water shortage and nitrogen deficiency were applied on a herbicide sensitive population to observe their influence on the plants. The sensitive plants presented significantly lower Fv/Fm values than the resistant plants 3 days after treatment (DAT) for the ALS and ACCase inhibitors. On the same day, and for the same treatments the Fv/Fm values of the resistant plants were not affected and similar to the control. Appling a PS II inhibitor reduced the Fv/Fm values of both sensitive and resistant plants rapidly. Yet, sensitive and resistant plants could clearly be separated on 4 DAT based on the different Fv/Fm values. On the other hand, nitrogen deficiency did not influence the photosystem II measurements. Water shortage reduced rapidly the Fv/Fm value of the plants seven days after the application, yet at this point plant symptoms included the death of the plants. According to this experiment, the Weed PAM® sensor has proved its capability to identify the sensitive and resistant A. myosuroides populations shortly after the herbicide application. Paper 2. A verification of the above results was made under field conditions for different A. myosuroides populations and different locations. On the first part 50 populations in total including both sensitive and herbicide resistant populations were tested. The second part field experiments were conducted in ten locations around Germany over two years with the local field population mix. It was investigated if the Weed PAM® sensor could separate between herbicide sensitive and resistant A. myosuroides populations 5 DAT. The different populations were sown in a winter wheat field. Two ACCase- and three ALS- inhibitors were applied. In all herbicide treatments, Fv/Fm values of A. myosuroides were significantly lower than the untreated plants at the 5 DAT. For each location, measurements were conducted at 5 DAT. A visual measurement, to verify the result, was carried out at 21 DAT. In both cases, 95% of the plants were correctly identified as sensitive or resistant. This demonstrated the ability of the Weed PAM® sensor to conduct in field real time detection of herbicide resistant A. myosuroides populations shortly after treatment. Paper 3. Greenhouse and field experiments were carried out to investigate if the chlorophyll fluorescence of soybean plants was altered, under herbicide stress. Herbicide combinations including inhibitors of PS II, DOXP synthase, cell division and microtubule assembly were selected for different pre-emergence treatments. Herbicide combinations including inhibitors of PS II, ALS and ACCase were applied in post-emergence treatments. Chlorophyll fluorescence was measured from the emergence of soybeans until the three/four-leaf stage. Furthermore the stress effect of the different treatments on the soybean plants was determined by measuring their dry biomass. In the greenhouse, post-emergence treatments with ALS and ACCase inhibitors did not seem to induce stress on the soybean plants. As expected, it originally demonstrated low Fv/Fm values when stressed by PS II inhibitors. But the PS II system recovered soon, one week after emergence. Stress induced by other pre-emergence herbicides occurred one week after emergence and lasted longer than the stress induced by the PS II inhibitors. Dry biomass collaborated with the sensor result. Based on the current thesis, the Weed PAM® system can be an important tool in the identification of herbicide resistant weed populations, in a timely manner. It has proven its capabilities both in A. myosuroides as a weed and in soybean plants. This technology will help farmers to take more suitable weed control strategies, as well as less economic and environmental risks.Die weltweite Zunahme an Herbizidresistenzen stellt eine der größten Herausforderungen der heutigen Unkrautbekämpfung dar. Die heutige Landwirtschaft verlangt eine effiziente, kostengünstige und zuverlässige Methode um Herbizidresistenzen an Unkräutern direkt im Feld zu erkennen. Hierzu wurden mehrere Studien im Gewächshaus und unter Feldbedingungen durchgeführt, die in drei Artikeln veröffentlicht wurden. Experiment 1. In diesem Teil der Arbeit wurde ein Gewächshausexperiment mit dem bildgebenden Chlorophyllfluoreszenz-Sensor Weed PAM® durchgeführt. Um die Effektivität von Herbiziden festzustellen wurde der vielversprechende Parameter Maximaler Photosystem II Quantenertrag (Fv/Fm) mit dem Sensor gemessen. Im ersten Versuch wurden sechs verschiedene Herbizide an sensitiven sowie resistenten Alopecurus myosuroides Populationen getestet. Im zweiten Versuch wurden sensitive Populationen Wasserknappheit und Stickstoffmangel ausgesetzt, um deren Stressreaktionen zu beobachten. Dieser Versuch trug dazu bei die Einflüsse von abiotischen Faktoren auf die Fv/Fm-Werte zu erkennen. Die Ergebnisse zeigten, dass 3 Tage nach einer Behandlung (TNB) mit ALS- und ACCase-Hemmern die sensitiven Pflanzen signifikant geringere Fv/Fm-Werte aufwiesen, als die resistenten Populationen. Es zeigte sich nur ein geringer Einfluss des Herbizidstresses auf das Photosystem II der resistenten Pflanzen nach der Behandlung mit ALS- und ACCase-Hemmern. Die Fv/Fm-Werte sensitiver und resistenter Pflanzen fielen unter dem Einfluss von PS II Hemmern jedoch rapide ab. 4 TNB zeigte sich, dass die Fv/Fm-Werte der beiden Populationen sich signifikant unterschieden. Stickstoffmangel hatte während der Messungen keinen signifikanten Einfluss auf das Photosystem II, wohingegen sieben Tage nach Initiierung der Wasserknappheit eine schnelle Reduktion der Fv/Fm-Werte aufgetreten ist. Nach den Ergebnissen dieses Experimentes ist der Weed PAM® Sensor dazu in der Lage kurz nach einer Herbizidbehandlung sensitive und resistente Populationen von A. myosuroides zu erkennen. Experiment 2. Dieses Experiment untersuchte die Erkennung von Herbizideffektivität auf sensitive Pflanzen unter Feldbedingungen mithilfe des Weed PAM® Sensors. Zudem wurde getestet, ob der Sensor in der Lage ist unter diesen Bedingungen auch herbizidresistente A.myosuroides Populationen 5 TNB festzustellen. Auf einem Winterweizenschlag wurde eine herbizidsensitive Population von A. myosuroides ausgesät und mit zwei ACCase- und drei ALS-Hemmern behandelt. Die Fv/Fm-Werte der A.myosuroides Pflanzen waren 5 TNB in allen Herbizidbehandlungen signifikant geringer im Vergleich zu den unbehandelten Pflanzen. Innerhalb von 2 Jahren wurden in einem weiteren Experiment insgesamt 50 sensitive und resistente Populationen an zehn Standorten getestet. Eine visuelle Bonitur erfolgte 21 TNB. Die Ergebnisse zeigten, dass 95% der Erkennungen korrekt durchgeführt wurden. Dies zeigt die hohe Genauigkeit des Weed PAM® Sensors für eine direkte Herbizidresistenz-Erkennung von A.myosuroides Populationen kurz nach der Applikation unter Feldbedingungen. Experiment 3. In diesem Teil der Arbeit wurden in Gewächshaus- und Feldversuchen Sojabohnen einem Herbizidstress ausgesetzt, um zu untersuchen, ob sich die Chlorophyllfluoreszenz-Emissionen nach einer Herbizidapplikation ändern. Herbizid-Kombinationen mit Hemmern des PS II-Systems, der DOXP Synthase und der Zellteilung, sowie des Mikrotubuliaufbaus stellten die Vorauflauf-Varianten dar. Die Nachauflauf-Varianten bestanden aus Herbizidmischungen mit PS II, ALS- und ACCase hemmern. Die Chlorophyllfluoreszenz wurde direkt nach dem Auflaufen der Sojabohnen bis zum 3-4 Blatt Stadium gemessen. Durch die Messung der Trockenmasse der Sojabohnen wurde das Stressniveau bestimmt. Im Gewächshausexperiment wurde beobachtet, dass kein Stress durch die Nachauflauf-Varianten in den Sojabohnen induziert wurde. Pflanzen, die durch PS II-Hemmer gestresst wurden, zeigten nach dem Auflaufen geringe Fv/Fm-Werte. Das Photosystem II der Pflanzen erholte sich jedoch innerhalb einer Woche auf das Niveau der unbehandelten Kontrolle. Der Stress durch andere Nachauflauf-Varianten trat eine Woche nach dem Auflauf auf und dauerte länger an als die Variante mit PS II-Hemmern. Die Aufnahmen der Trockenmasse bestätigten die Erkenntnisse der auf Chlorophyllfluoreszenz basierten Stresserkennung. Unter Zuhilfenahme des Weed PAM® Systems ist es Landwirten möglich die richtigen Unkrautbekämpfungsmaßnahmen noch in derselben Vegetationsperiode zu ergreifen in der das Resistenzproblem in ihrem Feld erkannt wurde. Zusammenfassend hilft diese neue Technologie den Landwirten geeignetere Unkrautbekämpfungsstrategien zu entwickeln und geringere ökonomische und ökologische Risiken einzugehen

Development of Herbicide Tolerant Tomato

Tomato is a major horticulture crop grown across the globe. Unfortunately, its yield is reduced by 25% because of auxin herbicides and glyphosate drift. In this present study, wild germplasm of tomato was screened for herbicide tolerance. From the greenhouse study nine accessions for glyphosate and 2,4-D, eleven accessions for dicamba, five accessions for quinclorac, eight accessions for aminocyclopyrachlor, and two accessions for picloram and aminopyralid were identified to be tolerant. A few accessions were selected from each herbicide tolerant group for field trials at two locations in Mississippi in 2016 and 2017. Results indicated that TOM18 was most tolerant to dicamba herbicide, while TOM87 and TOM129 to glyphosate and quinclorac herbicide, respectively, on the basis of yield and injury. Molecular experiments were conducted to measure the genetic diversity among diverse germplasm. Genetic diversity analysis showed wild accessions to be highly diverse as compared to cultivated tomato

Herbicides

Herbicides are one of the most widely used groups of pesticides worldwide for controlling weedy species in agricultural and non-crop settings. Due to the extensive use of herbicides and their value in weed management, herbicide research remains crucial for ensuring continued effective use of herbicides while minimizing detrimental effects to ecosystems. Presently, a wide range of research continues to focus on the physiology of herbicide action, the environmental impact of herbicides, and safety. The authors of Herbicides, Physiology of Action, and Safety cover multiple topics concerning current valuable herbicide research

Herbicide Resistance in Plants

Today, herbicide-resistant weeds dominate research and development efforts in the discipline of weed science. The incidence, management challenges, and cost of multiple herbicide-resistant weed populations are continually increasing worldwide. Crop varieties with multiple herbicide-resistance traits are being rapidly adopted by growers and land managers to keep ahead of the weed resistance tsunami. This Special Issue of Plants comprises papers that describe the current status and future outlook of herbicide resistance research and development in weedy and domestic plants, with topics covering the full spectrum from resistance mechanisms to resistance management. The unifying framework for this Special issue is the challenge posed to all of the contributing authors: What are the (potential) implications for herbicide resistance management

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

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

Investigating the potential for glyphosate resistance evolution in UK weedy species

Glyphosate is the world’s most used herbicide. There are currently 32 weedy species with resistant populations in 25 countries, although at present there are no reported cases of glyphosate resistance in the UK. As glyphosate use and selection pressure increases in the UK there is an excellent opportunity to investigate the potential for glyphosate resistance, and the evolutionary processes that may lead to resistance. The variability in standing genetic variation to herbicide susceptibility between weed populations can affect the amount of selection pressure and generations needed for resistance to evolve. If herbicide doses act within this standing genetic variation there may be a reduction in sensitivity due to a buildup of minor alleles related to reduced sensitivity. This thesis has investigated the glyphosate response of three UK weedy species, Alopecurus myosuroides (blackgrass), Anisantha sterilis (sterile brome), and Arabidopsis thaliana. Dose-response experiments showed significant variation in susceptibility between populations of all three species. Glasshouse selection experiments tested if glyphosate sensitivity could be further reduced under directional selection with below field rate doses, in Alopecurus myosuroides populations. Following selection, ten of eleven selected lines showed significantly different ED50 and ED90 values compared to unselected control lines, demonstrating that there is potential for selection of reduced glyphosate sensitivity, which may result in compromised field efficacy. Fitness cost experiments for two glyphosate-selected lines showed no major fitness costs associated with decreased glyphosate susceptibility both with and without wheat competition. Analysis of multi-parent advanced generation inter-cross Arabidopsis thaliana lines highlighted an area on chromosome 2 of the Arabidopsis thaliana genome that may be associated with variation in glyphosate susceptibility. These results are discussed in the context of the possibility of glyphosate resistance evolution in the UK

Physiological, and Genetic Characterization of 2,4-D-resistant Palmer Amaranth (Amaranthus palmeri S. Watson) and Its Management

Doctor of PhilosophyDepartment of AgronomyMithila JugulamPalmer amaranth (Amaranthus palmeri S. Watson) is one of the topmost troublesome, C4 dioecious weeds in the US. Biological traits such as aggressive growth habits, prolific seed production, and the ability to withstand environmental stresses hinder control of this weed. Additionally, numerous Palmer amaranth populations across the US have been found to have evolved resistance to multiple herbicides. In 2018, a population of Palmer amaranth from a conservation tillage study from Riley County, Kansas was suspected to have evolved resistance to multiple herbicides including 2,4-dichlorophenoxyacetic acid (2,4-D) and was designated as Kansas Conservation Tillage Resistant (KCTR). 2,4-D, a synthetic auxin herbicide, is widely used for controlling broadleaf weeds in cereal crops. However, over-reliance on 2,4-D to control other herbicide-resistant weeds, along with the commercialization of 2,4-D-tolerant crop technology, has resulted in increased usage of this herbicide. The objectives of this dissertation were to 1) characterize the evolution of multiple herbicide resistance including 2,4-D in KCTR Palmer amaranth; 2) investigate the physiological mechanism of 2,4-D resistance in KCTR compared to two known susceptible Palmer amaranth populations i.e., Kansas Susceptible (KSS) and Mississippi Susceptible (MSS); 3) assess the genetic basis of 2,4-D resistance in KCTR; and 4) evaluate herbicide programs that can manage glyphosate-resistant Palmer amaranth in 2,4-D tolerant soybean. Experiments were conducted under either greenhouse or controlled growth chamber conditions. Standard herbicide dose-response, physiological, biochemical (using radiolabeled herbicides), breeding, and field experiments were designed and conducted. The results of these experiments found that KCTR Palmer amaranth had evolved resistance to six herbicide modes of action, including acetolactate synthase (ALS)-, photosystem II (PS II)-, 5- enolpyruvylshikimate-3-phosphate synthase (EPSPS)-, 4-hydroxyphenylpyruvate dioxygenase (HPPD)-, protoporphyrinogen oxidase (PPO)- inhibitors, and synthetic auxins (2,4-D). Sequencing and analyses of genes coding for the herbicide targets indicated absence of all known mutations that confer resistance, except for EPSPS-inhibitor, with a massive amplification of EPSPS gene (up to 88 copies). Investigation of non-target site resistance mechanism(s) in KCTR confirmed the predominance of metabolic resistance to multiple herbicides mediated by either cytochrome P450 (P450) or glutathione S-transferase enzyme activity. Whole-plant doseresponse analyses confirmed a 6- to 11- fold resistance to 2,4-D in KCTR compared to two susceptible populations (KSS or MSS). [14C] 2,4-D uptake and translocation studies indicated a 10% less and 3 times slower translocation of [14C] 2,4-D in KCTR compared to susceptible populations, while there was no difference in the amount of [14C] 2,4-D absorbed. However, KCTR plants metabolized [14C] 2,4-D much faster than the susceptible KSS and MSS, suggesting that enhanced metabolism bestows resistance to this herbicide in KCTR. Further, use of P450-inhibitor (e.g., malathion) indicated that the metabolism of 2,4-D in KCTR is mediated by P450 activity. Genetic analyses of F1 and F2 progenies, derived from crossing between KCTR and KSS, revealed that 2,4-D resistance in KCTR Palmer amaranth is an incompletely dominant, nuclear trait. Segregation of F2 progenies did not follow the Mendelian single gene inheritance model (3:1), suggesting the involvement of multiple genes in mediating 2,4-D resistance in KCTR. Evaluation of herbicide programs for Palmer amaranth management in the field suggested that pre-emergence herbicides with residual activity followed by post-emergence application of either 2,4-D or glufosinate or 2,4-D and glufosinate can control glyphosateresistant Palmer amaranth in 2,4-D-tolerant soybean. Overall, the outcome of this dissertation documents the first case of a six-way resistance in a single Palmer amaranth population and also for the first time characterizes the physiological and genetic basis of 2,4-D resistance in this weed. These findings will help in predicting and minimizing further evolution and spread of 2,4- D resistance in Palmer amaranth