<b>Mutation of Trp-574-Leu ALS gene confers resistance of radish biotypes to iodosulfuron and imazethapyr herbicides

Abstract

ABSTRACT. Acetolactate synthase inhibitors are the main group of herbicides used in winter crops in Southern Brazil where their intensive use has selected for herbicide-resistant biotypes of radish. The resistance affects the efficacy of herbicides, and identifying the resistance mechanism involved is important for defining management strategies. The aim of this study was to elucidate the resistance mechanism of radish biotypes by quantifying the enzyme activity, ALS gene sequencing and evaluating the response of biotypes to iodosulfuron and imazethapyr herbicide application after treatment with a cytochrome P 450 monooxygenase inhibitor. The susceptible (B 1 ) and resistant (B 4 and B 13 ) biotypes were from wheat fields in the Northwest of Rio Grande do Sul State. The results demonstrated that the enzyme affinity for the substrate (K M ) was not affected in biotypes B 4 and B 13 but that the V max of the resistant biotypes was higher than that of biotype B 1 . The resistant biotypes showed no differential metabolic response to iodosulfuron and imazethapyr herbicides when inhibited by malathion and piperonyl butoxide. However, gene sequencing of ALS showed a mutation at position 574, with an amino acid substitution of tryptophan for leucine (Trp-574-Leu) in resistant biotypes.Keywords: Raphanus sativus, mechanism of resistence, ALS enzyme activity, gene mutation, metabolism.Mutação da Trp-574-Leu do gene ALS confere resistência de biótipos de nabo ao herbicida iodosulfurom e imazetapir RESUMO. Os inibidores da acetolactato sintase são o principal grupo de herbicidas usados em culturas de inverno do Sul do Brasil onde seu uso intenso selecionou biótipos resistentes de nabo. A resistência afeta a eficácia dos herbicidas, e a identificação do mecanismo de resistência envolvido é importante na definição de estratégias de manejo. O objetivo deste estudo foi elucidar o mecanismo de resistência em biótipos de nabo através da quantificação da atividade da enzima, sequenciamento do gene ALS e avaliar a resposta dos biótipos à aplicação do herbicida iodosulfurom e imazetapir após tratamento com inibidores do metabolismo da citocromo P 450 monooxigenase. Os biótipos suscetível (B 1 ) e resistentes (B 4 e B 13 ) eram de lavouras de trigo da região Noroeste do Estado do Rio Grande do Sul. Os resultados demonstraram que a afinidade da enzima pelo substrato (K M ) não foi afetada nos biótipos B 4 e B 13 , porém o V max dos biótipos resistentes foi superior quando comparado ao biótipo B 1 . Os biótipos resistentes não apresentaram resposta metabólica diferencial ao herbicida iodosulfurom e imazetapir quando inibidos pelo malathion e butóxido de piperolina. Entranto, o sequenciamento do gene ALS evidenciou mutação na posição 574 com uma substituição do aminoácido triptofano por leucina (Trp-574-Leu) nos biótipos resistentes. Palavras-chave:Raphanus sativus, mecanismo de resistência, atividade da enzima ALS, mutação gênica, metabolismo.

Target‐site resistance and cross‐resistance to ALS‐inhibiting herbicides in radish and wild radish biotypes from Brazil

“…The four resistant biotypes had a homozygous amino acid substitution of tryptophan to leucine (Trp574‐Leu; Table 4). This mutation belongs to Domain B of the ALS gene and is known to cause resistance to all chemical groups of ALS‐inhibiting herbicides in several weeds (Tranel et al., 2020), including wild radish (Tan & Medd, 2002) and radish (Cechin et al., 2017; Pandolfo et al., 2016). Radish biotypes with the Trp574‐Leu mutation presented high level of resistance to imidazolinone and sulfonylurea (Cechin et al., 2017; Pandolfo et al., 2016).…”

“…This mutation belongs to Domain B of the ALS gene and is known to cause resistance to all chemical groups of ALS‐inhibiting herbicides in several weeds (Tranel et al., 2020), including wild radish (Tan & Medd, 2002) and radish (Cechin et al., 2017; Pandolfo et al., 2016). Radish biotypes with the Trp574‐Leu mutation presented high level of resistance to imidazolinone and sulfonylurea (Cechin et al., 2017; Pandolfo et al., 2016). However, structural modification of the enzyme due to the point‐mutation of Trp574‐Leu can confer resistance to all chemical groups of ALS‐inhibiting herbicide (Duggleby et al., 2008).…”

“…These results indicated a comparatively lower resistance level than the resistant populations of radish and wild radish from Australia and Argentina, where the GR 50 values were greater than 640 for chlorsulfuron (Han et al., 2012) and more than 43 g ha −1 to metsulfuron‐methyl (Pandolfo et al., 2013). The use of high rates of herbicides to control ALS resistant plants have been reported for radish (Cechin et al., 2017), horseweed ( Conyza canadensis ; Zheng et al., 2011), and waterhemp ( Amaranthus tuberculatus ; Patzoldt & Tranel, 2007). Pandolfo et al.…”

“…the ALS inhibiting herbicides of the imidazolinone chemical group, where a high concentration greater than 200 μM was needed to inhibit 50% the ALS enzyme activity in radish biotypes (Cechin et al, 2017;Pandolfo et al, 2016). The resistance level to different ALS-inhibiting herbicides differs according to the mutation based on the specificity of the bind-ing of the herbicide molecule to the action site (Yu & Powles, 2014;Yu et al, 2012).…”

“…The results showed a high level of resistance to imazapic, with RF greater than 175 for the JUC2 and CRA3 (radish) and CAF3 (wild radish) biotypes, and intermediate level of resistance for the CAF2 biotype (Table 3). Similarly, mutation of Try-574-Leu confer high levels of resistance in radish T A B L E 3 Amount of herbicide required to inhibit 50% of enzyme activity (I 50 ) with 95% confidence intervals (CI), and resistance factors (RF) in resistant radish (JUC2 and CRA3) and resistant wild radish (CAF2 and CAF3) biotypes subjected to different rates of imazapic herbicide biotypes to imazethapyr, and intermediate level of resistance to iodosulfuron-methyl-sodium (Cechin et al, 2017) and metsulfuron-methyl herbicide (Pandolfo et al, 2016). According to these authors, the substitution of tryptophan to leucine amino acid in the 574 position of ALS gene confers higher levels of resistance to imidazolinone and cross-resistance to ALS-inhibiting herbicides.…”