京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第12881号 / 論農博第2808号 / 新制||農||1028(附属図書館) / 学位論文||H26||N4880(農学部図書室) / 31599 / (主査)教授 冨永 達, 教授 奥本 裕, 教授 宮川 恒 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Muscarinic acetylcholine receptors (mAChRs) are G-protein-coupled receptors that are underutilized for controlling insect pests despite their involvement in various physiological functions. To-date, there are no commercialized insecticides targeting insect mAChRs. In this dissertation, effective target-site synergism was demonstrated in susceptible Drosophila melanogaster where mAChR agonism by pilocarpine enhanced the toxicity of insecticides targeting gamma-aminobutyric acid (GABA)-gated chloride channels, indicating the potential of insect mAChRs as a target for developing novel insecticides/synergists to control resistant pests. A point mutation (A301S) in the GABA-gated chloride channel confers resistance to dieldrin (Rdl), lindane, and fipronil, which I have confirmed using different routes of exposure. However, the same synergistic effect was not achieved in the resistant strain with the presence of this target-site mutation. This difference between two strains is perplexing because there is a change in the efficacy of several compound classes that do not directly act upon GABA-gated chloride channels. Specifically, a point mutation appears to influence how the insect central nervous system (CNS) responds to muscarinic compounds, type I pyrethroids, and acetylcholinesterase (AChE) inhibitors. In the case of acetylcholinesterase, the resistant insect increases the expression of Ace gene encoding this enzyme. Fully understanding how the CNS responds to receptor modifications is not well understood and could have a significant impact to pest management strategies. / Doctor of Philosophy / Insects significantly influence the food production, health, and the economy of the human world. Control of insect pest outbreaks relies on the proper use of insecticides. However, extensive application of insecticides has resulted in pests being able to adapt to these compounds, through insecticide resistance. Ultimately, this will affect currently used pest management strategies. To help alleviate this urgent problem, my dissertation provided an alternative strategy to control pests, which is to use a mixture of two molecules that influence different targets in the insect nervous system that could reduce the use of toxic or deleterious compounds that are the active ingredients. It is important to not solely rely on current insecticides on the market and find new insecticides that work differently. I used the fruit fly to help me understand how insecticide mixtures would work, but also understand how the complex nature of insect adaptations at the level of the nervous system continues to threaten pest management. Based on studies that were performed here, we now have a better understanding on how to investigate the failure of insecticides in the field, which will ultimately help us make new molecules.

L’afide del pesco Myzus persicae rappresenta uno degli insetti più dannosi in agricoltura. Estremamente polifago e cosmopolita, viene combattuto principalmente con trattamenti insetticidi. Nel corso degli ultimi anni neonicotinoidi e piretroidi hanno rappresentato i componenti principali delle strategie di difesa contro questa specie, ma recenti programmi di monitoraggio condotti in Sud Europa hanno rivelato la presenza di popolazioni resistenti, mettendo in dubbio l’efficacia a lungo termine di queste classi di prodotti. Il presente lavoro prende in esame la diffusione dei principali meccanismi di resistenza agli insetticidi nelle popolazioni di M. persicae presenti sul territorio italiano. La prima parte si concentra sulle resistenze target-site e considera la distribuzione delle principali mutazioni che sono state associate alla resistenza a neonicotinoidi e piretroidi. La seconda parte riguarda le resistenze metaboliche e analizza le principali classi di enzimi associate ad attività di sequestro o detossificazione delle molecole di insetticida. Infine, il progetto si focalizza sulla caratterizzazione delle possibili interazioni tra tali enzimi detossificanti e molecole sinergizzanti quali il ben noto piperonil butossido (PBO). I risultati ottenuti consentiranno di migliorare le strategie di difesa per evitare trattamenti inefficaci e mantenere il più a lungo possibile l’efficacia dei prodotti oggi disponibili per il controllo di M. persicae. / The green peach aphid Myzus persicae is a globally significant crop pest, controlled mainly by chemical treatments. In recent years neonicotinoids and pyrethroids have been the main components of pest management strategies used by growers. However, recent monitoring programmes in Southern Europe have shown the widespread presence of resistant populations, posing a serious threat to the long-term efficacy of these insecticide classes. The present work aims to characterise the main biochemical and molecular mechanisms responsible for insecticide resistance in Italian populations of M. Persicae. The first part is focused on target-site resistance and consider the frequency and distribution of the main target-site mutations associated with neonicotinoid and pyrethroid resistance. The second part is related to metabolic resistance and analyses the involvement of detoxifying enzymes able to sequester or metabolise the insecticide molecules. Furthermore, the project aims to characterise possible interactions between those enzymes and synergistic compounds like the well-known piperonyl butoxide (PBO). Results obtained by this investigation will help to improve insecticide resistance management strategies, in order to avoid ineffective applications and maintain the long-term sustainability of chemical control against M. persicae.

A planta daninha Amaranthus palmeri é nativa dos Estados Unidos, porém foi pela primeira vez relatada no Brasil no ano de 2015. Embora comprovadamente com resistência múltipla aos herbicidas inibidores da ALS e da EPSPS, até o momento não foram investigadas as bases moleculares da resistência. Além disso, por causa da recente introdução da planta daninha no país, alternativas de manejo com culturas tolerantes a herbicidas necessitam ser estudadas. Sendo assim, os objetivos desse trabalho são de caracterizar a espécie de planta daninha introduzida no país, identificar os mecanismos de resistência aos herbicidas inibidores da ALS e da EPSPS presentes no biótipo, e propor abordagens de manejo em ambientes dos novos eventos transgênicos resistentes a herbicidas. Um bioensaio utilizando marcadores genéticos foi desenvolvido para confirmar que a população coletada no estado do Mato Grosso (BR-R) é A. palmeri, e não A. tuberculatus, outra espécie dióica do gênero Amaranthus. Os resultados de experimentos de curvas de dose-resposta e acúmulo de chiquimato indicaram que a BR-R possui alto nível de resistência, com DL50 de 4.426 e 3.400 g glyphosate ha-1 no primeiro e segundo experimento, respectivamente, mais que o dobro da dose típicamente recomendada para o controle da espécie e, adicionalmente, observou se acúmulo mínimo de chiquimato a concentração de 1 mM nos tecidos das plantas tratadas com o herbicida. BR-R também foi resistente a herbicidas dos grupos químicos das sulfoniluréias e imidazolinonas. O mecanismo de resistência ao glyphosate encontrado nesta população foi a super expressão gência, através do aumento no número de cópias do gene da EPSPS no genoma da planta BR-R, entre 50 e 179 cópias adicionais. Além disso, duas substituições de aminoácidos foram observadas na sequência da ALS, W574L e S653N, conferindo resistência tanto a sulfoniluréias quanto a imidazolinonas. No experimento utilizandos os herbicidas correspondentes às culturas geneticamente modificadas com novos traits de tolerância a herbicidas observou se, de uma forma geral, que as associações de herbicidas apresentaram níveis de controle mais satisfatórios. Assim, esta pesquisa confirma a introdução de da espécie A. palmeri no Brasil, assim como a resistência múltipla aos herbicidas inibidores da EPSPS e da ALS. Seu manejo é mais eficaz através da associação de herbicidas, garantindo assim o uso racional das novas tecnologias de culturas geneticamente modificadas com tolerância a herbicidas. / Palmer Amaranth (Amaranthus palmeri) is a weed species native to the United States, but it was reported in Brazil for the first time in 2015. Despite this population being resistant to EPSPS and ALS inhibitors, the molecular basis of its multiple resistance is unknown up to date. Because of this species introduction to Brazil, alternatives of management with the new herbicide-tolerant crops technologies need to be studied. The objectives of this research are to characterize the weed species introduced to Brazil, identify the mechanisms conferring resistance to ALS and EPSPS inhibitors herbicides, and to propose management approaches in environments with the new genetically modified herbicide-tolerant crops. A genotyping bioassay using genetic markers was developed to confirm that the species collected in the state of Mato Grosso (BR-R) is indeed A. palmeri and not A. tuberculatus, another dioceous species in the Amaranthus genus. Dose-response experiments and shikimate accumulation bioassay data indicate high level of resistance, with LD50 of 4,426 and 3,400 g glyphosate ha-1 in the first and second experiments, respectively, higher than the double rate tipically recommended to control it, and minimal accumulation in BR-R with 1 mM of glyphosate in treated plants in the leaf disks assay. BR-R also was resistanto to sulfonilurea and imidazolinone herbicides. The mechanism conferring resistance to glyphosate identified in this population was gene amplification, with increased EPSPS copy number - between 50 and 179 more copies in BR-R. Besides, two target-site mutations were identified in the ALS gene sequencing, W574L and S653N, conferring resistance to sulfonilureas and imidazolinones. The weed control experiment, overal, herbicide tank mixtures achieved higher levels of control. Therefore, this research confirms the introduction of A. palmeri to Brazil, as well as its multiple resistance to EPSPS and ALS inhibitor herbicides. Its control is more efficient with herbicide mixtures, which guarantees more susteinable use of the new herbicide-tolerant crop technologies.

A planta daninha Amaranthus palmeri é nativa dos Estados Unidos, porém foi pela primeira vez relatada no Brasil no ano de 2015. Embora comprovadamente com resistência múltipla aos herbicidas inibidores da ALS e da EPSPS, até o momento não foram investigadas as bases moleculares da resistência. Além disso, por causa da recente introdução da planta daninha no país, alternativas de manejo com culturas tolerantes a herbicidas necessitam ser estudadas. Sendo assim, os objetivos desse trabalho são de caracterizar a espécie de planta daninha introduzida no país, identificar os mecanismos de resistência aos herbicidas inibidores da ALS e da EPSPS presentes no biótipo, e propor abordagens de manejo em ambientes dos novos eventos transgênicos resistentes a herbicidas. Um bioensaio utilizando marcadores genéticos foi desenvolvido para confirmar que a população coletada no estado do Mato Grosso (BR-R) é A. palmeri, e não A. tuberculatus, outra espécie dióica do gênero Amaranthus. Os resultados de experimentos de curvas de dose-resposta e acúmulo de chiquimato indicaram que a BR-R possui alto nível de resistência, com DL50 de 4.426 e 3.400 g glyphosate ha-1 no primeiro e segundo experimento, respectivamente, mais que o dobro da dose típicamente recomendada para o controle da espécie e, adicionalmente, observou se acúmulo mínimo de chiquimato a concentração de 1 mM nos tecidos das plantas tratadas com o herbicida. BR-R também foi resistente a herbicidas dos grupos químicos das sulfoniluréias e imidazolinonas. O mecanismo de resistência ao glyphosate encontrado nesta população foi a super expressão gência, através do aumento no número de cópias do gene da EPSPS no genoma da planta BR-R, entre 50 e 179 cópias adicionais. Além disso, duas substituições de aminoácidos foram observadas na sequência da ALS, W574L e S653N, conferindo resistência tanto a sulfoniluréias quanto a imidazolinonas. No experimento utilizandos os herbicidas correspondentes às culturas geneticamente modificadas com novos traits de tolerância a herbicidas observou se, de uma forma geral, que as associações de herbicidas apresentaram níveis de controle mais satisfatórios. Assim, esta pesquisa confirma a introdução de da espécie A. palmeri no Brasil, assim como a resistência múltipla aos herbicidas inibidores da EPSPS e da ALS. Seu manejo é mais eficaz através da associação de herbicidas, garantindo assim o uso racional das novas tecnologias de culturas geneticamente modificadas com tolerância a herbicidas. / Palmer Amaranth (Amaranthus palmeri) is a weed species native to the United States, but it was reported in Brazil for the first time in 2015. Despite this population being resistant to EPSPS and ALS inhibitors, the molecular basis of its multiple resistance is unknown up to date. Because of this species introduction to Brazil, alternatives of management with the new herbicide-tolerant crops technologies need to be studied. The objectives of this research are to characterize the weed species introduced to Brazil, identify the mechanisms conferring resistance to ALS and EPSPS inhibitors herbicides, and to propose management approaches in environments with the new genetically modified herbicide-tolerant crops. A genotyping bioassay using genetic markers was developed to confirm that the species collected in the state of Mato Grosso (BR-R) is indeed A. palmeri and not A. tuberculatus, another dioceous species in the Amaranthus genus. Dose-response experiments and shikimate accumulation bioassay data indicate high level of resistance, with LD50 of 4,426 and 3,400 g glyphosate ha-1 in the first and second experiments, respectively, higher than the double rate tipically recommended to control it, and minimal accumulation in BR-R with 1 mM of glyphosate in treated plants in the leaf disks assay. BR-R also was resistanto to sulfonilurea and imidazolinone herbicides. The mechanism conferring resistance to glyphosate identified in this population was gene amplification, with increased EPSPS copy number - between 50 and 179 more copies in BR-R. Besides, two target-site mutations were identified in the ALS gene sequencing, W574L and S653N, conferring resistance to sulfonilureas and imidazolinones. The weed control experiment, overal, herbicide tank mixtures achieved higher levels of control. Therefore, this research confirms the introduction of A. palmeri to Brazil, as well as its multiple resistance to EPSPS and ALS inhibitor herbicides. Its control is more efficient with herbicide mixtures, which guarantees more susteinable use of the new herbicide-tolerant crop technologies.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第17830号 / 農博第2015号 / 新制||農||1016(附属図書館) / 学位論文||H25||N4787(農学部図書室) / 30645 / 京都大学大学院農学研究科農学専攻 / (主査)教授 稲村 達也, 教授 冨永 達, 教授 奥本 裕 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

No description available.

The first case of field evolved acetolactate synthase (ALS) inhibiting herbicide resistance in the model plant, mouse-ear cress, was reported in winter wheat fields in Westmoreland County, Virginia. A putative resistant (R) mouse-ear population was assessed for ALS resistance relative to a putative susceptible (S) and a susceptible lab population Columbia (C). Results indicated that the R population needed 23 to >2400 fold rate of thifensulfuron relative to S or C population, and it has evolved cross-resistance to sulfonylureas (SU), triazolopyrimidine sulfonanilides (TP), and sulfonylaminocarbonyltriazolinones (SCT). Further studies sequenced the whole genome for four field populations, representing two locations and two resistance levels (high and low) per location, to characterize the genetic mechanism of ALS resistance. The results revealed that all populations contained mutations in the ALS gene at the Pro197 site, although the Pro was substituted by Phe in one location and Thr in the other. Also, both high- and low-level resistant plants at one location had additional mutations (Trp574Leu or Asp376Glu) known to confer resistance to ALS inhibiting herbicides. Patterns of herbicide cross-resistance also varied among the populations. Additionally, research was conducted to assess preemergent (PRE) and postemergent (POST) alternative herbicide options for control of ALS resistant mouse-ear cress and its interference with winter wheat. Results indicate flumioxazin, pyroxasulfone, and metribuzin can be used for effective PRE control whereas 2,4-D, dicamba, and metribuzin can be effective post control options. No mouse-ear cress interference with winter wheat was observed at density of more than 300 plants m-2. / Master of Science in Life Sciences / The first case of field evolved acetolactate synthase (ALS) inhibiting herbicide resistance in mouse-ear cress, was reported in winter wheat fields in Westmoreland County, Virginia. A putative resistant (R) mouse-ear population was assessed for ALS resistance relative to a putative susceptible (S) and a susceptible lab population Columbia (C). The ALS resistance was confirmed in greenhouse and the R population exhibited cross-resistance to three ALS herbicide chemical families. Further studies sequenced the whole genome for four field populations collected from Essex and Westmoreland Counties, Virginia to characterize the genetic mechanism of ALS resistance. The results revealed that all populations contained target site mutations. All populations had a mutation at a commonly implicated point within ALS gene; however, substitutions varied by location. Populations from one location had multiple target site mutation contrary to populations from second location which had only one mutation. Patterns of ALS cross-resistance also varied among the populations. Additionally, research was conducted to assess preemergent (PRE) and postemergent (POST) alternative herbicide options for control of ALS resistant mouse-ear cress, and its interference with winter wheat. Results indicate flumioxazin, pyroxasulfone, and metribuzin can be used for effective PRE control whereas 2,4-D, dicamba, and metribuzin can be effective post control options. No wheat yield loss was observed from mouse-ear cress interference at a density of more than 300 plants m⁻².

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22080号 / 農博第2372号 / 新制||農||1072(附属図書館) / 学位論文||R1||N5234(農学部図書室) / 京都大学大学院農学研究科農学専攻 / (主査)教授 冨永 達, 教授 奥本 裕, 教授 白岩 立彦 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Sustainable weed control in managed agricultural systems requires the judicious use of multiple weed control tactics and prevents over-reliance on any one tactic. In this context, sustainable weed management plays a critical role in the mitigation of one of agriculture's most pressing problems- herbicide resistance. Research conducted in Virginia sought to explore the effects of integrating multiple weed management tactics in corn and cool-season turfgrass. Additionally, research was conducted to confirm nicosulfuron and glyphosate herbicide resistance in Virginia johnsongrass and elucidate the molecular mechanisms conferring those resistances. Rye and hairy vetch cover crop residues, combined with reduced rates of preemergence herbicide and postemergence glyphosate applications, were shown to provide sufficient weed control and corn yield. Cover crop type or residue level did not augment weed control in corn production systems, but the use of glyphosate was essential for late-season weed control. Rye and vetch biculture as a cover crop increased corn yield compared to rye cover crop alone. In cool-season turfgrass, the addition of reduced preemergence herbicide rates to corn gluten meal, an organic herbicide product, reduced crabgrass 25%. Moreover, control was dependent on herbicide choice. Herbicides applied at half of recommended labeled rates or less did not control crabgrass at a commercially-acceptable level, regardless of corn gluten meal addition. In field experiments, Virginia johnsongrass expressed resistance to nicosulfuron and glyphosate. Glyphosate at 0.88 kg ae ha-1 controlled johnsongrass 65%. Nicosulfuron at 0.14 kg ai ha-1 controlled the same population 10%. Greenhouse experiments confirmed differential sensitivity of putative herbicide-resistant johnsongrass seedlings to nicosulfuron and glyphosate when compared to a susceptible population. Herbicide resistance was not conferred via target-site mutation. Five ALS-gene site mutations were confirmed absent in Virginia johnsongrass, while three others were located in coding regions that could not be elucidated in johnsongrass. Further investigations showed glyphosate resistance was not conferred via reduction in herbicide absorption or translocation. The susceptible johnsongrass caused an increase in a polar metabolite at Rf = 0.17 with concomitant reduction in glyphosate over time. Although the mechanism is not clear, these data suggests that glyphosate resistance in johnsongrass may be associated with differential metabolism. / Ph. D.