Molecular bases for resistance to acetyl-coenzyme A carboxylase inhibitor in Japanese foxtail (Alopecurus japonicus)

BACKGROUND: Haloxyfop‐R‐methyl is a widely used herbicide to control Poaceae weeds. Alopecurus japonicus, a widespread annual grass, can no longer be controlled by haloxyfop‐R‐methyl after continuous use of this herbicide for several years. RESULTS: Dose‐response experiments have established that the Js‐R biotype of A. japonicas has evolved resistance to aryloxyphenoxypropionates (APPs). Target‐site enzyme sensitivity experiments have established that the haloxyfop (free acid) rate causing 50% inhibition of acetyl‐CoA carboxylase (ACCase) activity (I₅₀) for the resistant (Js‐R) biotype is 11 times higher than that for the susceptible (Js‐S) biotype. In many cases, resistance to ACCase‐inhibiting herbicides is due to a resistant ACCase enzyme. Full‐length DNA and mRNA sequences of the plastidic ACCase gene were amplified. Eight single‐nucleotide differences were detected in this region. Four of the nucleotide changes were silent mutations. However, the other four nucleotide mutations caused four amino acid substitutions, replacing Arg‐1734 with Gly, Met‐1738 with Leu, Thr‐1739 with Ser and Ile‐2041 with Asn in the R biotype respectively; the substitution at position 2041 had been reported, while the other three had not. CONCLUSION: The ACCase in the Js‐R biotype was less susceptible to haloxyfop‐R‐methyl than that in the Js‐S biotype. Moreover, the amino acid substitution of Ile‐2041 with Asn might confer resistance to haloxyfop‐R‐methyl in A. japonicas. Copyright © 2012 Society of Chemical Industry     

安徽省部分地区冬小麦田日本看麦娘Alopecurus japonicus对精噁唑禾草灵的抗性发生现状及ACCase基因突变研究

日本看麦娘Alopecurus japonicus是中国冬小麦田和油菜田主要恶性禾本科杂草之一。为了明确安徽省部分地区日本看麦娘对精齅唑禾草灵抗性发生情况及可能存在的抗性机制,本研究在安徽省天长市日本看麦娘发生严重区域冬小麦田共采集10个种群,采用温室盆栽法在整株水平上测定了不同种群对精齅唑禾草灵的抗性水平,扩增并比对了抗性和敏感种群之间靶标酶乙酰辅酶A羧化酶 (Acetyl-CoA carboxylase,ACCase) 基因部分序列的差异。结果显示,与敏感种群相比,10个抗性种群对精齅唑禾草灵均产生了高水平抗性,抗性指数在30.50~58.55之间。不同抗性种群均发生了ACCase基因突变,其中8个种群发生了第1 781位异亮氨酸 (Ile) 到亮氨酸 (Leu) 突变,2个种群发生了第2 027位色氨酸 (Trp) 到半胱氨酸 (Cys) 突变。此外,各种群均具有较高的ACCase基因突变频率 (≥80%)。研究表明,抗性日本看麦娘在安徽省部分地区发生较为严重,ACCase基因突变是导致不同日本看麦娘种群对精齅唑禾草灵产生抗性的重要原因之一。相对于第2 027位,日本看麦娘ACCase基因更倾向于在第1 781位产生突变以表现靶标抗性。     

Amino acid substitution in Ace paralogous acetylcholinesterase accompanied by organophosphate resistance in the spider mite Tetranychus kanzawai

Insecticide resistant strains of the kanzawa spider mite, Tetranychus kanzawai, with insensitive AChE have spread widely throughout Japan. To clarify the molecular mechanism of this insensitivity, acetylcholinesterase (AChE) cDNA of the resistant strains of T. kanzawai was determined based on the AChE cDNA sequence of Tetranychus urticae and the sequences compared between the two spider mite species. The cDNA encoded 687 amino acids of AChE primary structure showing high homology to T. urticae. Amino acid homology indicated that the AChE is an Ace paralogous type of insect AChE. There were only three substitutions of amino acid residues between the AChEs of the two species. In the AChE of the resistant strain of T. kanzawai, one of the three amino acid substitutions was Phe439Trp, which lines the acyl pocket of the enzyme active site. Considering that the same substitution was found at the equivalent position of Ace paralogous AChE in the resistant strain of Culex tritaeniorhynchus, Phe439Trp substitution likely plays an important role in the insecticide insensitivity of the mite AChE.     

Characterization of imidacloprid resistance in the housefly Musca domestica (Diptera: Muscidae)

In recent years, imidacloprid was introduced to control the housefly in China and it was documented that the housefly indeed showed signs of resistance to imidacloprid somewhere but not in China. Therefore, a housefly population collected from filed (IFS) was selected continuously with imidacloprid to establish the resistant strain (IRS) and the basic characteristics were investigated in this study. After continuous selection over 21 generations, the resistance ratio increased from 9.01 to 140, and different levels of cross-resistance were developed to beta-cypermethrin, chlorpyrifos, chlorfenapyr, acetamiprid and azamethiphos in the IRS strain. The realized heritability of resistance was 0.10. The synergistic ratios for IRS pretreated with DEF, DEM and PBO were 1.68, 1.52 and 2.53, and the corresponding ones for IFS were 3.17, 1.87 and 2.67, respectively. Synergistic and biochemical assays suggested that the cytochrome P450 may play an important role in the imidacloprid resistance comparing with GSTs- and carboxylesterases-mediated detoxification in the IRS strain, and there might be additional mechanisms (e.g. reduced target-site sensitivity) contributed to imidacloprid resistance in the IRS strain.     

抗精噁唑禾草灵的日本看麦娘ACCase基因突变

为明确日本看麦娘抗性种群对精噁唑禾草灵的抗性水平及抗性产生的分子机制,采用整株水平测定法测定了日本看麦娘对精噁唑禾草灵的抗性水平,扩增和比对了日本看麦娘抗性和敏感种群间乙酰辅酶A羧化酶(acetyl-Co A carboxylase,ACCase)基因的差异。结果显示,与敏感种群AH-7相比,抗性种群AH-25对精噁唑禾草灵的抗性倍数为33.82;AH-25种群ACCase基因CT区域2 078位氨基酸发生了突变,由天冬氨酸GAT突变为甘氨酸GGT;AH-25种群对炔草酯、烯草酮和烯禾啶产生了高水平的抗性,抗性倍数分别为35.66、38.64和29.14,对高效氟吡甲禾灵产生了低水平的抗性,抗性倍数为3.04,对精喹禾灵和唑啉草酯较敏感。表明ACCase基因2 078位氨基酸的突变可能是导致精噁唑禾草灵产生高水平抗性的重要原因。     

Mechanism of resistance to quinclorac in smooth crabgrass (Digitaria ischaemum)

The mechanism of resistance to quinclorac was investigated in a smooth crabgrass biotype [Digitaria ischaemum (Schreb. ex Schweig) Schreb. ex Muhl] from Tulare County, California. Quinclorac (8.96 kg a.i. ha⁻¹) had no effect (P = 0.18) on the resistant (R) biotype, but reduced fresh weight of a susceptible (S) biotype by 93%. After treatment with 4.48 kg a.i. quinclorac ha⁻¹, the S biotype produced about three times more ethylene than the R biotype and accumulated cyanide in tissues. Similar amounts of endogenous cyanide resulting from treatment with KCN reproduced quinclorac phytotoxicity. Pre-treatment with the ACC synthase inhibitor AVG reduced quinclorac phytotoxicity by 37% and ethylene production by 89%. These data suggest a target site-based mechanism of resistance involving stimulation of ACC synthesis and accumulation of cyanide. Also, the R biotype had four times more β-cyanoalanine synthase activity than the S biotype, suggesting a higher ability to detoxify cyanide.     

Target-site basis for resistance to acetolactate synthase inhibitor in Water chickweed (Myosoton aquaticum L.)

Water chickweed is a widespread and competitive winter annual or biennial weed of wheat in China. One Water chickweed population (HN02) resistant to several acetolactate synthase (ALS) inhibitors was found in Henan province of China. Whole-plant bioassays showed that HN02 was high resistance to tribenuron (292.05-flod). In vitro ALS assays revealed that resistance was due to reduced sensitivity of the ALS enzyme to tribenuron. The I₅₀ value for HN02 was 85.53 times greater respectively than that of susceptible population (SD05). This altered ALS sensitivity in the resistant population was due to a mutation in the ALS gene resulting in a Pro197 to Ser substitution. Cross-resistance experiments indicated that HN02 exhibited various resistance patterns to pyrithiobac-sodium, florasulam and pyroxsulam, without resistance to imazethapyr. This is the first report of tribenuron-resistant Water chickweed in Henan province of China, target-site based resistance was established as being due to an insensitive form of ALS, resulting from a Pro to Ser substitution at amino acid position 197 in the ALS gene.     

Cross-resistance to dicyclanil in cyromazine-resistant mutants of Drosophila melanogaster and Lucilia cuprina

Cyromazine is an insect growth regulator insecticide with a novel mode of action, mainly used to control dipteran insects. Previously, cyromazine-resistant mutants of the Australian sheep blowfly Lucilia cuprina and the vinegar fly Drosophila melanogaster have been isolated following ethyl methanesulfonate mutagenesis and selection for resistance. Here, we show that these cyromazime-resistant mutants are cross-resistant to dicyclanil, an insect growth regulator compound with a similar chemical structure to cyromazine. Dicyclanil was recently introduced as a control agent of L. cuprina. Cross-resistance to the benzylphenylurea insecticide lufenuron was also assessed. Only one D. melanogaster cyromazine-resistant mutant is cross-resistant to lufenuron.     

Indoxacarb resistance in the house fly, Musca domestica

Indoxacarb (DPX-MP062) is a recently introduced oxadiazine insecticide with activity against a wide range of pests, including house flies. It is metabolically decarbomethoxylated to DCJW. Selection of field collected house flies with indoxacarb produced a New York indoxacarb-resistant (NYINDR) strain with >118-fold resistance after three generations. Resistance in NYINDR could be partially overcome with the P450 inhibitor piperonyl butoxide (PBO), but the synergists diethyl maleate and S,S,S-tributyl phosphorothioate did not alter expression of the resistance, suggesting P450 monooxygenases, but not esterases or glutathione S-transferases are involved in the indoxacarb resistance. Conversely, the NYINDR strain showed only 3.2-fold resistance to DCJW, and this resistance could be suppressed with PBO. Only limited levels of cross-resistance were detected to pyrethroid, organophosphate, carbamate or chlorinated hydrocarbon insecticides in NYINDR. Indoxacarb resistance in the NYINDR strain was inherited primarily as a completely recessive trait. Analysis of the phenotypes vs. mortality data revealed that the major factor for indoxacarb resistance is located on autosome 4 with a minor factor on autosome 3. It appears these genes have not previously been associated with insecticide resistance.     

Triazophos resistance mechanisms in the rice stem borer (Chilo suppressalis Walker)

A field population of the rice stem borer (Chilo suppressalis Walker) with 203.3-fold resistance to triazophos was collected. After 8-generation of continuous selection with triazophos in laboratory, resistance increased to 787.2-fold, and at the same time, the resistance to isocarbophos and methamidophos was also enhanced by 1.9- and 1.4-fold, respectively, implying some cross-resistance between triazophos and these two organophosphate insecticides. Resistance to abamectin was slightly enhanced by triazophos selection, and fipronil and methomyl decreased. Synergism experiments in vivo with TPP, PBO, and DEM were performed to gain a potential indication of roles of detoxicating enzymes in triazophos resistance. The synergism results revealed that TPP (SR, 1.92) and PBO (SR 1.63) had significant synergistic effects on triazophos in resistant rice borers. While DEM (SR 0.83) showed no effects. Assays of enzyme activity in vitro demonstrated that the resistant strain had higher activity of esterase and microsomal O-demethylase than the susceptible strain (1.20- and 1.30-fold, respectively). For glutathione S-transferase activity, no difference was found between the resistant and the susceptible strain when DCNB was used as substrate. However, 1.28-fold higher activity was observed in the resistant strain when CDNB was used. These results showed that esterase and microsomal-O-demethylase play some roles in the resistance. Some iso-enzyme of glutathione S-transferase may involve in the resistance to other insecticides, for this resistant strain was selected from a field population with multiple resistance background. Acetylcholinesterase as the triazophos target was also compared. The results revealed significant differences between the resistant and susceptible strain. The V_max and K_m of the enzyme in resistant strain was only 32 and 65% that in the susceptible strain, respectively. Inhibition tests in vitro showed that I₅₀ of triazophos on AChE of the resistant strain was 2.52-fold higher. Therefore, insensitive AChE may also involved in triazophos resistance mechanism of rice stem borer.     

Selection and characterization of spinosad resistance in Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae)

The cross-resistance and biochemical mechanism of the beet armyworm, Spodoptera exigua (Hübner), to spinosad was studied in the laboratory. S. exigua population were collected from Shanghai suburb. After five generations of selection, the resistance of S. exigua to spinosad increased 345.4 times compared with the susceptible strain. There was no cross-resistance between spinosad and fenvalerate, phoxim, methomyl, abamectin, and cyfluthrin. When the inhibitors, PBO, TPP, DEF, and DEM were used as synergist in the susceptible strain and resistant strain, the synergistic ratio was 0.7-, 0.5-, 1.0-, and 0.6- fold for the susceptible strain, and 9.8-, 1.5-, 2.6-, and 1.5-fold for the resistant strain, respectively. The results revealed that PBO had significant synergistic effect on the resistant strain. The activity in vitro of microsomal-O-demethylase and glutathione S-transferase in the resistant strain was 5.2- and 1.0-fold of the susceptible strain, respectively. The results implied that microsomal-O-demethylase might be important in conferring spinosad resistance in the S. exigua population.     

The role of cytochrome P450 monooxygenase in the different responses to fenoxaprop-P-ethyl in annual bluegrass (Poa annua L.) and short awned foxtail (Alopecurus aequalis Sobol.)

Herbicide resistance or tolerance in weeds mediated by cytochrome P450 monooxygenase is a considerable problem. However, cytochrome P450 mediated resistance or tolerance in weeds was less studied. Thus, in this work, the role of the cytochrome P450 monooxygenase in the different responses of Poa annua and Alopecurus aequalis to fenoxaprop-P-ethyl was studied. We found that the effect of fenoxaprop-P-ethyl could be synergized by piperonyl butoxide (PBO) in P. annua, but not by malathion. After being treated with fenoxaprop-P-ethyl (containing mefenpyr-diethyl), the contents of cytochrome P450 and cytochrome b₅ in P. annua increased significantly compared to plants treated with mefenpyr-diethyl only or untreated plants. However, the increase was less in A. aequalis, which was susceptible to fenoxaprop-P-ethyl. The activities of ρ-nitroanisole O-demethylase (PNOD), ethoxyresorufin O-deethylase (EROD), ethoxycoumarin oxidase (ECOD) and NADPH-dependent cytochrome P450 reductase mediated by cytochrome P450 monooxygenase increased in P. annua after treatment with fenoxaprop-P-ethyl, especially the activities of ECOD and cytochrome P450 reductase. Besides this, cytochrome P450 monooxygenase activity toward fenoxaprop-P-ethyl in P. annua increased significantly compared to untreated or treated with mefenpyr-diethyl plants and treated or untreated A. aequalis. Cytochrome P450 monooxygenase may play an important role in the different responses to fenoxaprop-P-ethyl in P. annua and A. aequalis.     

A mutation confers Monochoria vaginalis resistance to sulfonylureas that target acetolactate synthase

Acetolactate synthase (ALS) is the target enzyme for four distinct families of compounds: sulfonylureas (SUs), imidazolinones, triazolopyrimidine sulfonanilides, and pyrimidinyl oxybenzoates. We cloned and sequenced the fragments encoding ALS genes from biotypes of Monochoria vaginalis susceptible (S) and resistant (R) to SU-herbicides. The nucleotide sequences of the 39 bp Domain A region for R M. vaginalis biotype differed from that of the S biotype by a single nucleotide substitution at variable Pro codon of Domain A (CCT to TCT), predicting a Pro in the S but a Ser in the R biotype. No nucleotide differences between S and R M. vaginalis were observed in Domain D. We suggest that the amino acid substitution at Domain A region is responsible for resistance to SU-herbicides in M. vaginalis collected from Ushiku City, Ibaraki Prefecture, Japan.     

Reduced paraquat translocation in paraquat resistant Arctotheca calendula (L.) Levyns is a consequence of the primary resistance mechanism, not the cause

Resistance to paraquat has been studied in detail in many weed species for more than a decade, with the precise mechanism of resistance still unclear. Several studies have indicated that reduced movement of the herbicide to the site of action in the chloroplast is at least partly responsible for endowing resistance. Although paraquat translocation studies have been performed in the past it has been rare for these studies to have been conducted on whole plants in the light, despite early observations which clearly showed that paraquat translocation is minimal unless treated plants are exposed to light. This study has addressed this issue in Arctotheca calendula by tracing the movement of ¹⁴C-paraquat in resistant and susceptible plants in both the dark and light. Differences in paraquat translocation between the resistant and susceptible biotypes of A. calendula were only observed when treated plants were exposed to light. It was observed that paraquat translocation was significantly reduced in the resistant compared to the susceptible biotype when plants were exposed to light but not in the dark. It is postulated that paraquat translocation is dependent on light mediated damage. As paraquat-induced damage is less severe in paraquat resistant plants, overall paraquat translocation is reduced in the resistant biotype.     

Possible involvement of herbicide sequestration in the resistance to diclofop-methyl in Italian biotypes of Lolium spp.

The mechanism of resistance to diclofop-methyl in three Italian populations of Lolium spp. (two resistant and one susceptible) was investigated. The major proportion of R-1 (Tuscania 1997) and R-2 (Roma 1994) plants (approximately 80%) survived after herbicide treatment by emitting new tillers from the crown. Both resistant (R-1 and R-2) and susceptible (Vetralla 1994) Lolium spp. populations were target-site sensitive. No difference in diclofop-methyl absorption by shoots of resistant and susceptible biotypes was observed. At the dose corresponding to 1× the recommended field rate, a relatively higher metabolism was found in R-2 biotype. In contrast, at the doses 2× and 10× the field rate no difference in herbicide metabolism between susceptible and resistant biotypes was observed. At all the three herbicide doses (1×, 2×, and 10× the field rate) 48 h after the treatment (HAT), the total amount of metabolites produced by wheat was more than three times higher than that produced by resistant and susceptible ryegrass biotypes. At the doses 1× and 2× the field rate, the herbicide translocation was different in the susceptible biotypes compared to resistant biotypes. The total amount of the radiolabel found 48 HAT in culm and root was approximately twice in susceptible biotype than in resistant biotypes. Susceptible and resistant ryegrass biotypes differed in the capability of their roots to acidify the external medium. Susceptible biotype acidified the external solution at approximately 6 times the rates of the resistant biotypes. In the present study, the mechanism responsible for resistance in the investigated resistant biotypes was not univocally identified. Indirect evidence supports the possible involvement of herbicide sequestration or immobilization.     

山东省小麦田播娘蒿对苯磺隆的抗性测定

为明确山东省冬小麦田播娘蒿对苯磺隆的抗性水平及分布现状,在温室中采用整株剂量-反应测定法测定了山东省各小麦主产区的37份播娘蒿对苯磺隆的敏感性。结果显示,山东省播娘蒿对苯磺隆的抗药性已非常普遍,所测样品中,抗性生物型有29个,占总采样数的78.38%,敏感生物型仅8个,占21.62%。抗性生物型中,中抗性生物型最多占51.72%,低抗性生物型占31.03%,高抗性生物型占17.24%。鲁西南平洼区和鲁西北平原区播娘蒿抗性水平普遍较高,鲁南山区、胶潍河谷平原区和鲁北滨海区抗性水平相对较低。     

Molecular basis of resistance to sulfonylureas in Papaver rhoeas

A Papaver rhoeas population resistant to several acetolactate synthase (ALS) inhibiting herbicides, called 25/98, was found in Catalonia (Northeastern of Spain). This population has an altered form of the enzyme that showed cross-resistance to several herbicides of this group. The highest resistance was found with tribenuron-methyl and sulfometuron-methyl. Studies were conducted to define the molecular basis of this resistance. Two regions of the ALS gene were amplified using degenerated universal primers and sequenced. Population 25/98 contained a single nucleotide substitution in domain A changing Pro₁₉₇ by Ser (using the nomenclature of Arabidopsis thaliana) that confers sulfonylurea resistance. Another change was detected in a region located outside of any conserved domains described to date, but its implication in the resistance remains unclear. We analyze the putative role of the found mutations in relation to the observed resistance using a putative three-dimensional model of the Papaver ALS enzyme.     

Monitoring of beet armyworm Spodoptera exigua (Lepidoptera: Noctuidae) resistance to chlorantraniliprole in China

Beet armyworm, Spodoptera exigua is a major insect pest of vegetables in China, and has been reported to develop resistance to many broad-spectrum insecticides. Recently registered chlorantraniliprole provides a novel option for control of this pest resistant to other conventional insecticides. The susceptibilities of field collected populations were measured by diet incorporation assay with neonate, obvious variation of susceptibility was observed among the 18 field populations with LC₅₀ values varying from 0.039 to 0.240 mg/liter. Moderate resistant level was discovered in 8 of 18 field populations, other 8 populations had become low level tolerance to chlorantraniliprole, and only one population in all the field colonies remained susceptible. Biochemical assays were performed to determine the potential mechanisms involved in tolerance variation. Field populations displayed varied detoxification enzyme activities, but the regression analysis between chlorantraniliprole toxicities and enzyme activities demonstrated each field population might have specific biochemical mechanisms for tolerance. Artificial selection in laboratory with chlorantraniliprole was carried out, 23 generations of continuous selections resulted in 11.8-fold increase in resistance to chlorantraniliprole, and 3.0-fold and 3.7-fold increases in mixed function oxidase and esterase, respectively. Compared with the susceptible strain kept in laboratory the selection strain had developed 128.6-fold resistance to this insecticide. Synergism assays showed the detoxification enzymes might not involved in the resistance observed in field collected populations and the selected strain.     

甘蓝型油菜对核盘菌及其毒素的抗性遗传分析

采用Griffing双列杂交第四类遗传试验设计,运用朱军发展的加性-显性遗传模型,直接估算了甘蓝型油菜抗核盘菌及其毒素草酸的遗传方差、遗传力和基因效应.抗病性鉴定采用温室病圃和草酸浸根鉴定法,它们分别鉴定了对核盘菌和草酸的抗性.结果表明,油菜对核盘菌及草酸的抗性加性方差和显性方差均极显著(P＜0.01),抗病性主要由加性和显性基因控制,且对核盘菌抗性的加性方差大于显性方差,而对草酸抗性则是显性方差大于加性方差.油菜对核盘菌和草酸的广义遗传力分别为0.750和0.576,狭义遗传力分别为0.403和0.236.高遗传力表明可通过适当的抗病性鉴定方法有效地培育抗病品种(系).基因效应评价结果表明,抗、感亲本的基因效应是不同的,其中抗病亲本783-3具有较理想的加性效应值,同时在多数组合中有较低的显性效应值,是抗病育种的优良亲本,而感病亲本相反.抗×感病的后代既可能为抗病,也可能为感病.