Purification,characterization and comparison of glutathione S-transferases from black-grass (Alopecurus myosuroides Huds) biotypes

In the UK biotypes of black-grass (Alopecurus myosuroides Huds) showing resistance to both chlorotoluron (CTU) and aryloxyphenoxypropionate graminicides are increasingly being observed. Although the precise mechanisms involved in this resistance have yet to be identified, increased herbicide metabolism has been implicated as being involved in at least some cases of resistance. Glutathione S-transferases (GSTs) are a group of enzymes which have been demonstrated to metabolise herbicides in some plants, and the resistant black-grass biotype Peldon contains approximately double the GST activity towards 1-chloro-2,4-dinitrobenzene (CDNB) of susceptible biotypes. To investigate further the possible role of GSTs in herbicide resistance in black-grass, a purification procedure has been developed for these enzymes. A 27.5 kDa polypeptide possessing GST activity was purified from the susceptible biotype Herbiseed. Purification of GSTs from the resistant biotype Peldon also identified this polypeptide along with an additional 30 kDa polypeptide. An in-vitro kinetic study of both crude and purified GST extracts, and western blot analysis using antisera raised against the 27.5 kDa polypeptide, suggest that the 30 kDa polypeptide may possess GST activity, and is not a precursor of the 27.5 kDa polypeptide. These results are discussed and compared to GST profiles for other weeds and crops demonstrating herbicide resistance or tolerance. © 1999 Society of Chemical Industry     

Developmental changes in glutathione S‐transferase activity in herbicide‐resistant populations of Alopecurus myosuroides Huds (black‐grass) in the field

Herbicide‐resistant populations of Alopecurus myosuroides Huds (black‐grass) have become widespread throughout the UK since the early 1980s. Clear evidence suggests that more than one resistance mechanism exists, and glutathione S‐transferases (GSTs) have been implicated in resistance due to enhanced metabolism. This study reports the determination of GST activity in four UK black‐grass populations from field sites situated in the East Midlands. Data demonstrate that, as untreated plants in the field mature, there is an accompanying natural elevation of GST activity with natural environmental changes from winter to spring. We speculate that this endogenous change in enzyme activity with plant development in the field contributes to reduced efficacy of some graminicides applied in the spring. These observations are discussed in relation to predicting herbicide efficacy to achieve maximum control of this important grass weed. © 2001 Society of Chemical Industry     

Glutathione transferases in herbicide-resistant and herbicide-susceptible black-grass (Alopecurus myosuroides)

Glutathione transferase (GST) activities toward the selective herbicide fenoxaprop-ethyl, together with thiol contents, have been compared in seedlings of wheat (Triticum aestivum) and two populations of black-grass (Alopecurus myosuroides) which are resistant to a range of herbicides (Peldon and Lincs E1), and a black-grass population which is susceptible to herbicides (Rothamsted). GST activities toward the non-cereal herbicides metolachlor and fluorodifen were also determined. On the basis of enzyme specific activity, GST activities toward fenoxaprop-ethyl in the leaves were in the order wheat>Peldon=Lincs E1>Rothamsted, while with fluorodifen and metolachlor the order was Peldon=Lincs E1>Rothamsted>wheat. Using an antibody raised to the major GST from wheat, which is composed of 25-kDa subunits, it was shown that the enhanced GST activities in both Peldon and Lincs E1 correlated with an increased expression of a 25-kDa polypeptide and the appearance of novel 27-kDa and 28-kDa polypeptides. Leaves of both wheat and black-grass contained glutathione and hydroxymethylglutathione, with the concentrations of glutathione being in the order Peldon>Lincs E1=Rothamsted=wheat. However, in glasshouse dose-response assays, the Lincs E1 population showed much greater resistance to fenoxaprop-ethyl than Peldon. We conclude that high GST activities and the availability of glutathione may contribute partially to the relative tolerance of black-grass to herbicides detoxified by glutathione conjugation. Although herbicide-resistant populations show enhanced GST expression, in the case of fenoxaprop-ethyl the associated increased detoxifying activities alone cannot explain the differences between populations in the degree of resistance seen at the whole plant level. ©1997 SCI     

New, quick tests for herbicide resistance in black-grass (Alopecurus myosuroides Huds) based on increased glutathione S-transferase activity and abundance

Black-grass (Alopecurus myosuroides Huds) is a major grass weed in winter cereals in Europe. It reduces yields and can act as a secondary host for a range of diseases. Herbicide resistance in this species was first detected in the UK in the early 1980s, and has now been reported in thirty counties. To successfully manage herbicide resistance it is vital that suspect populations are tested so that appropriate action can be taken. Ideally, a test will be quick, cheap and easy to use. Furthermore, it should provide an unequivocal result before post-emergence herbicides are to be applied, allowing alternative strategies to be adopted where necessary. This paper reports the development of new tests for herbicide resistance based on our observation that the resistant black-grass biotype Peldon contains approximately double the activity of the enzyme glutathione S-transferase (GST) compared with susceptible biotypes. Data are presented on the production of a monoclonal antiserum to a novel 30 kDa GST polypeptide purified from the biotype Peldon. An ELISA using this antiserum is described and the utility of this assay to detect resistant black-grass biotypes in plants grown under glass and in the field is presented. In addition, a microtitre assay for GST activity is described, which allows the rapid assessment of GST activities of plants. Both abundance and activity of GSTs are discussed as markers for herbicide resistance in black-grass.     

Mechanisms of resistance to simazine in Sonchus oleraceus

Sonchus oleraceus biotypes resistant to simazine were identified by chlorophyll fluorescence analysis of intact leaves. The mechanisms of resistance were determined on the basis of cpDNA and glutathione S‐transferase (GST) activity analyses. The results of the cpDNA analysis showed that most of the resistant biotypes had a Ser 264 Gly mutation in the psbA gene, which is responsible for an amino acid substitution in the D‐1 protein sequence. The cpDNA fragments were amplified by polymerase chain reaction and digested with restriction enzyme MaeI. Two restriction bands of 338 and 75 bp were recorded in biotypes with the target mutation, while three bands (218, 120 and 75 bp) were present in biotypes without this mutation. A second mechanism of resistance in this species was through the detoxification of simazine by conjugation with glutathione. In resistant biotypes without the above‐mentioned mutation, the average level of GST (simazine) activity in leaves was 4.5‐fold greater than in the resistant biotypes with the target mutation and 8.3‐fold greater than in the susceptible biotypes. Resistance as a result of the target mutation was more common than that achieved through detoxification by glutathione conjugation of simazine.     

Identification and expression pattern of a glutathione S‐transferase in Echinochloa crus‐galli

Plant glutathione S‐transferase (GST) forms a major part of the herbicide detoxification enzyme network in plants. A GST cDNA was isolated from Echinochloa crus‐galli and characterised. The gene, designated EcGST1 (E. crus‐galli GeneBank no: JX518596 ), has a 684 bp open reading frame predicted to encode a 25 kD protein. Sequence alignment showed that EcGST1 is a GST homologue. Its expression in response to quinclorac treatment was monitored in seedlings (leaves and roots) and adult plants (leaves, roots, stems and seeds) of quinclorac‐resistant (R) and susceptible (S) biotypes of E. crus‐galli. EcGST1 expression was 1.5–3 times greater in the R plants than in the S plants. However, after exposure to quinclorac, the difference in the expression levels of EcGST1 in R plants, compared with S plants, increased to a ratio of 6–10. Enhanced EcGST1 levels should enable greater quinclorac detoxification following quinclorac stimulation in R plants. GST‐based metabolism may be partially responsible for resistance to quinclorac in E. crus‐galli. The results suggest a new resistance mechanism for this R biotype in Chinese rice fields.     

Triazine herbicide resistance in Chenopodium album L.: Occurrence and characteristics of an intermediate biotype

Intermediate (I) biotypes for triazine herbicide resistance in Chenopodium album (as defined by a peculiar fluorescence curve), had the same ID₅₀ values as resistant(R) plants for chloroplast response to atrazine, but proved to be more susceptible at lower doses. Furthermore, the lethal dose in seedling treatments was lower than that of the R plants, but six times higher than for susceptible (S) plants. These I characteristics of I biotypes were maternally inherited in crosses. I biotypes were isolated from various progenies of susceptible precursor (Sp) plants in two garden populations. This could be the first step in the occurrence of triazine herbicide resistance. However, Sp plants have not been observed in field populations. The significance of the presence of a single isozyme pattern for all Sp plants is discussed. The results suggest an evolutionary pathway from S to R plants via I biotypes.     

The role of glutathione S-transferases in the detoxification of some organophosphorus insecticides in larvae and pupae of the yellow mealworm,Tenebrio molitor (Coleoptera: Tenebrionidae)

The correlation between the natural levels of glutathione S-transferase (GST) and the tolerance to the organophosphorus insecticides parathion-methyl and paraoxon-methyl, as well as the interaction of affinity-purified enzyme and the insecticides were investigated in order to collect further information on the role of the glutathione S-transferase system as a mechanism of defence against insecticides in insects. The studies were carried out on the larvae and pupae of the coleopteran Tenebrio molitor L, which exhibit varying natural levels of GST activity. Stage-dependent susceptibility of the insect against insecticides was observed during the first 24 h. However, 48 h after treatment, the KD₅₀ value increased significantly due to the recovery of some individuals. Simultaneous injection of insecticide with compounds which inhibit GST activity in vitro caused an alteration in susceptibility of insects 24 or 48 h post-treatment, depending on stage and insecticide used. Inhibition studies combined with competitive fluorescence spectroscopy revealed that the insecticides probably bind to the active site of the enzyme, thus inhibiting its activity towards 1-chloro-2,4-dinitrobenzene in a competitive manner. High-performance liquid chromatography and gas chromatography revealed that T molitor GST catalyses the conjugation of the insecticides studied to a reduced form of glutathione (GSH). From the above experimental results, it is considered that GST offers a protection against the organophosphorus insecticides studied by active site binding and subsequent conjugation with GSH. © 2001 Society of Chemical Industry     

Influence of temperature on the activity of AC 222,293 against Avena fatua L. and Alopecurus myosuroides Huds.

Foliage applications of AC 222,293 were more active against Alopecurus myosuroides Huds. plants growing at high (26/16°C day/night) compared to either medium (16/10°C) or low (11/7°C) temperatures. Soil activity was unaffected by temperature. Enhanced activity against A. myosuroides required exposure to the high temperatures for only 24 h immediately post spraying and correlated with the accumulation of greater levels of the biologically active acid in the apical meristem region during this period. Both foliage and soil activity of AC 222,293 were greatest against Avena fatua L. plants growing at medium (16/10°C) compared to either high (26/16°C) or low (11/7°C) temperatures. Foliar activity decreased slowly with increasing periods of high temperatures post spraying and then only at doses lower than those recommended for field use. Reduced performance at the high temperatures correlated with lower levels of the biologically active acid in the meristem region, despite greater uptake and translocation. This was due to increased metabolism of the parent herbicide and formation of metabolites other than the free acid.     

Non-enzymatic conjugation of fenoxaprop-ethyl with glutathione and cysteine in several grass species

Laboratory studies have shown that the amounts of glutathione (GSH) and cysteine are higher in grass species that are moderately tolerant, such as wheat (Triticum aestivum L., cv. Fredrick), and moderately susceptible, such as barley (Hor deum vitlgare L., cv. Legér) and triticale (cv. OAC Trillium), to fenoxaprop-ethyl (FE) than in species that are very susceptible to the her bicide, such as oat (Avena saliva L., cv. OAC Woodstock), wild oat (Avena fatua L.), yellow foxtail (Setaria glanca (L.) Bcauv.), large crab grass (Digitaria sanguinalis (L.) Scop.) and bar nyard grass (Echinochloa crus-galli (L.) P.B.). The safener, fenchlorazole-ethyl (FCE) was found to increase and decrease, respectively, the amounts of GSH and cysteine in the moderately tolerant and moderately susceptible species but had no effect on the susceptible species. It is sug gested that in the moderately tolerant and moderately susceptible species, especially following FCE treatment, more GSH is available to detoxify the herbicide. Glutathione-S-tranferase activity (GST) for FE was found to be very low in all of the species tested. In vitro experiments at physio-logical pH. demonstrated that FE may conjugate with GSH nonenzymatically. Therefore, it is suggested that nonenzymatic conjugation of fenoxaprop-ethyl with glutathione may be an important mechanism for tolerance of some grasses to this herbicide.     

Age-related mechanisms of propanil tolerance in Jungle-rice,Echinochloa colona

Uptake and metabolism of propanil were measured in both susceptible (S) and resistant (R) biotypes of Jungle-rice, Echinochloa colona (L.) link at different growth stages. Results showed that there was no significant difference in uptake between S and R biotypes of E. colona at any given growth stage, but that uptake was significantly reduced at older plant growth stages in all biotypes studied. Metabolism of propanil was more rapid in R biotypes than in S biotypes at all growth stages studied. Specific and total aryl acylamidase activity, responsible for the first stage of propanil metabolism, was higher in R biotypes than in S at all growth stages, but declined to about 50% of the maximum at older growth stages, confirming the importance of this enzyme in conferring resistance to this herbicide. The area of necrosis that developed around a single drop of propanil deposited on the adaxial leaf surface was used to assess the degree of propanil resistance; it was found that resistance increased at older E. colona growth stages in contrast to the rate of propanil metabolism and amidase activity. Treatment of leaves with the amidase inhibitors, carbaryl or piperophos, simultaneously with propanil, caused a decrease in resistance at growth stages where amidase activity was greatest. This treatment was less effective at older growth stages. These results show that, in E. colona, propanil metabolism is important for conferring resistance in younger plants (four-six-leaf stage). It is suggested that restricted uptake confers resistance in older plants.     

Negative cross-resistance to bentazone and pyridate in atrazine-resistant Amaranthus cruentus and Amaranthus hybridus biotypes

Plants of Amaranthus cruentus and Amaranthus hybridus resistant to atrazine and cyanazine were found in maize fields in north-eastern Spain. Both resistant foiotypes survived doses of 5 kg ha^?1 of atrazine and 2–4 kg ha^?1 of cyanazine but were controlled by lower doses of bentazone and pyridate than were susceptible biotypes. Such a negative cross-resistance was not found for chloroacetamides and MCPA. Chlorophyll fluorescence studies revealed that atrazine, bentazone, cyanazine and pyridate (10 mg litre^?1) caused inhibition of photosynlhetic electron transport in susceptible leaves, while in resistant plants, atrazine and cyanazine had no effect. Conversely, bentazone and pyridate inhibited photosynthesis to a greater extent in resistant than in susceptible biotypes. Isolated chloroplast membranes from resistant biotypes showed resistance factors of 366 and 501 to atrazine and 39 and 60 to cyanazine for A. hybridus and A. cruentus, respectively. Bentazone and pyridate were found to be more effective in chloropiasts of the resistant biotypes than those of the susceptible plants. It is suggested that enhanced susceptibility to bentazone and pyridate in triazine-resistant A. cruentus and A. hybridus biotypes may be associated with the alteration of the D-I polypeptide subunit of photosystem II, as found in triazine-resistant plants.     

Glutathione transferase activities toward herbicides used selectively in soybean

Using extracts from suspension-cultured cells of soybean (Glycine max cv. Mandarin) as a source of active enzymes, the activities of glutathione transferases (GSTs) catalysing the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) and selective herbicides were determined to be in the order CDNB≫ fomesafen>metolachlor=acifluorfen>chlorimuron-ethyl. GST activities showed a thiol dependence in a substrate-specific manner. Thus, GST activities toward acifluorfen and fomesafen were greater when homoglutathione (hGSH), the endogenously occurring thiol in soybean, was used as the co-substrate rather than glutathione (GSH). Compared with GSH, hGSH addition either reduced or had no effect on GST activities toward other substrates. In the absence of enzyme, the rates of hGSH conjugation with acifluorfen, chlorimuron-ethyl and fomesafen were negligible, suggesting that rapid hGSH conjugation in soybean must be catalysed by GSTs. GST activities were subsequently determined in 14-day-old plants of soybean and a number of annual grass and broadleaf weeds. GST activities of the plants were then related to observed sensitivities to post-emergence applications of the four herbicides. When enzyme activity was expressed on a mg^-1 protein basis, all grass weeds and Abutilon theophrasticontained considerably higher GST activity toward CDNB than soybean. With fomesafen as the substrate, GST activities were determined to be in the order soybean≫Echinochloa crus-galli>Digitaria sanguinalis>Sorghum halepense=Setaria faberi with none of the broadleaf weeds showing any activity. This order related well to the observed selectivity of fomesafen, with the exception of A. theophrasti, which was partially tolerant to the herbicide. Using metolachlor as the substrate the order of the GST activities was soybean>A. theophrasti≫S. halepense>Amaranthus retroflexus>Ipomoea hederacea, with the remaining species showing no activity. GST activities toward metolachlor correlated well with the selectivity of the herbicide toward the broadleaf weeds but not toward the grass weeds. Acifluorfen and chlorimuron-ethyl were selectively active on these species, but GST activities toward these herbicides could not be detected in crude extracts from whole plants. © 1997 SCI     

Effect of herbicide antidotes on glutathione content and glutathione S-transferase activity of sorghum shoots

The effects of the herbicide antidotes CGA-92194 (α-[(1,3-dioxolan-2-yl-methoxy)-imino]benzeneacetonitrile), flurazole [phenylmethyl 2-chloro-4-(trifluoromethyl)-5-thiazolecarboxylate], dichlormid (2,2-dichloro-N,N-di-2-propenylacetamide), and naphthalic anhydride (1H,3H-naphtho(1,8-cd)-pyran-1,3-dione) on nonprotein thiol content, glutathione content, and glutathione S-transferase (GST) activity in etiolated sorghum (Sorghum bicolor L.) Moench) shoots were examined. CGA-92194 and naphthalic anhydride had no effect on nonprotein thiol or reduced glutathione (GSH) content of sorghum shoots. In contrast, dichlormid and flurazole increased nonprotein thiol content of sorghum shoots by 24 and 48%, respectively. These increases were largely attributable to an increase in GSH. The antidotes increased GST activity less than twofold when using CDNB (1-chloro-2,4-dinitrobenzene) as a substrate. In contrast, when using metolachlor [2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl)acetamide] as a substrate, the increase in GST activity in response to antidote treatment was much greater: flurazole (30-fold), CGA-92194 (20-fold), naphthalic anhydride (17-fold), dichlormid (5-fold). The degree of protection from metolachlor injury conferred by a particular antidote was strongly correlated (R² = 0.95) with its ability to enhance GST activity, as evaluated with metolachlor as substrate. A comparison of GST activity in untreated and CGA-92194-treated seedlings, over a range of metolachlor concentrations (0.5–500 μM), indicated that the relative enhancement of enzyme activity by CGA-92194 was greater at lower metolachlor concentrations. The rate of nonenzymatic conjugation of metolachlor and GSH in vitro was much less (on a gram fresh weight equivalent basis) than the enzymatic rate. These results are consistent with the hypothesis that the above antidotes protect sorghum by enhancing GST activity which results in accelerated detoxification of metolachlor via GSH conjugation.     

Chlorotoluron Metabolism in Leaves of Resistant and Susceptible Biotypes of the Grass Weed Alopecurus myosuroides

The metabolism of the herbicide chlorotoluron by susceptible and resistant biotypes of the grass weed, Alopecurus myosuroides, was examined. After administration of radiolabelled herbicide to leaves, metabolites were extracted and analysed. The metabolites identified consisted of mono-demethylated-, di-demethylated- and ring methyl-hydroxylated chlorotoluron. Metabolism was more extensive in the resistant biotype, yielding principally the non-phytotoxic ring methyl-hydroxylated metabolite. The metabolites observed are characteristic of the activity of cytochrome P450 mixed-function oxygenase action. The specific cytochrome P450 inhibitor, 1-aminobenzotriazole, reduced accumulation of the ring methyl-hydroxylated metabolite in the resistant biotype.     

Status of black grass (Alopecurus myosuroides) resistance to acetyl-coenzyme A carboxylase inhibitors in France

We assessed the contributions of target site‐ and non‐target site‐based resistance to herbicides inhibiting acetyl‐coenzyme A carboxylase (ACC) in Alopecurus myosuroides (black grass). A total of 243 A. myosuroides populations collected across France were analysed using herbicide sensitivity bioassay (24 300 seedlings analysed) and ACC genotyping (13 188 seedlings analysed). Seedlings resistant to at least one ACC‐inhibiting herbicide were detected in 99.2% of the populations. Mutant, resistant ACC allele(s) were detected in 56.8% of the populations. Among the five resistant ACC alleles known in A. myosuroides, alleles containing an isoleucine‐to‐leucine substitution at codon 1781 were predominant (59.5% of the plants containing resistant ACC alleles). Comparison of the results from herbicide sensitivity bioassays with genotyping indicated that more than 75% of the plants resistant to ACC‐inhibiting herbicides in France would be resistant via increased herbicide metabolism. Analysis of herbicide application records suggested that in 15.9% of the populations studied, metabolism‐based resistance to ACC‐inhibiting herbicides was mostly selected for by herbicides with other modes of action. Our study revealed the importance of non‐target site‐based resistance in A. myosuroides. Using herbicides with alternative modes of action to control populations resistant to ACC‐inhibiting herbicides, the recommended management approach, may thus be jeopardised by the widespread occurrence of metabolism‐based resistance mechanisms conferring broad‐spectrum cross‐resistance.     

Propanil‐resistant barnyardgrass [Echinochloa crus‐galli (L.) Beauv.] in Sri Lanka: Seedling growth under different temperatures and control

Experiments were conducted to (i) evaluate the efficacy of propanil formulations available in Sri Lanka in controlling Echinochloa crus‐galli; (ii) study the seedling growth of propanil‐resistant (R) and ‐susceptible (S) biotypes of the weed under different temperatures; (iii) quantify the level of resistance in R biotypes and; (iv) to suggest alternative control measures for R biotypes. Field studies showed that retail propanil formulations (36% a.i., EC) applied at 2.7 kg a.i. ha^?1 gave less than 30% control of E. crus‐galli collected from several locations of the north dry zone of Sri Lanka. Chemical analysis revealed that there was no adulteration of propanil formulations at the retailer level. Growth studies conducted in controlled environments indicated that per cent germination and seedling growth of R and S biotypes were similar at the day/night temperature regimes imposed. However, per cent germination for plants grown under a 34/31°C (day/night) regime was 27–29% higher compared to those grown at 28/24°C. At the higher temperature regime, R and S biotypes reached the 2–3 leaf stage five days earlier, and the 4–5 leaf stage seven days earlier. The ED₅₀ values from the dose–response experiments indicated that the R biotype was four times more resistant to propanil than susceptible ones. The resistance index (RI) did not vary significantly under different temperature regimes. Quinclorac (25% a.i., SC) applied at 200 g a.i. ha^?1 and bispyribac‐sodium (10% a.i., SC) applied at 30 g a.i. ha^?1 (recommended dosages) successfully controlled propanil‐resistant biotypes of E. crus‐galli. Conversely, oxadiazon and propanil (8% and 23% a.i., EC, respectively) applied at 280 + 805 g a.i. ha^?1 did not result in satisfactory control.     

Comparaison de la germination et de la croissance de biotypes sensibles et résistants aux triazines chez quatre espèces de mauvaises herbes

Comparison of germination and growth of biotypes sensitive and resistant to triazines in four weed species. By observing germination under various conditions and plant development in non-competitive conditions, a comparison was made between sensitive and resistant biotypes of four species in which resistant populations have been discovered: Chenopodium album, Amaranthus retroflexus. Solanum nigrum and Polygonum lapathifolium. In spite of différent levels of significance, results indicate that the sensitive plants develop better. However, these findings may be modified according to the growth conditions. Moreover, high variance values in the characteristics measured reflect considerable heterogeneity at least in the sensitive lots. Nevertheless, seed from resistant plants of P. lapathifolium and, to a lesser extent, of Amaranthus, germinate more readily at low temperatures. In the light of these findings the authors discuss the possible advantages conferred on different lots by these characteristics, stressing the need to consider each species separately and to take into account the nature of the genotypes being compared. From such findings, while the exact factor determining resistance is not known, it is difficult to deduce a lower level of adaptability in resistant, as opposed to sensitive individuals, based on their place of origin and in the absence of herbicide treatments.     

Molecular basis of resistance to acetolactate synthase-inhibiting herbicides in Sisymbrium orientale and Brassica tournefortii

Three Australian Sisymbrium orientale and one Brassica tournefortii biotypes are resistant to acetolactate synthase (ALS)-inhibiting herbicides due to their possession of an ALS enzyme with decreased sensitivity to these herbicides. Enzyme kinetic studies revealed no interbiotypic differences within species in K_m (pyruvate) (the substrate concentration at which the reaction rate is half maximal) but a greater V_max (the rate when the enzyme is fully saturated with substrate) for two of the resistant S orientale biotypes over susceptible levels. F₁ hybrids from reciprocal crosses between resistant and susceptible biotypes of S orientale showed an intermediate response to chlorsulfuron compared to the parental plants. ALS herbicide resistance in S orientale segregated in a 3:1 (resistant:susceptible) ratio in F₂ plants with a single rate of chlorsulfuron, indicating that resistance is inherited as a single, incompletely dominant nuclear gene. Two regions of the ALS structural gene known to vary in ALS-resistant biotypes were amplified and sequenced. Resistant S orientale biotypes NS01 and SS03 contained a single nucleotide substitution in Domain B, predicting a Trp (in susceptible) to Leu (in resistant) amino acid change. Two adjacent nucleotide substitutions (CC T to AT T) predicting a Pro (in susceptible) to Ile (in resistant) change in the primary amino acid sequence were identified in Domain A of resistant S orientale biotype SS01. Likewise, a single nucleotide substitution at the same site in the resistant B tournefortii biotype predicts a Pro (in susceptible) to Ala (in resistant) substitution. No other interbiotypic nucleotide differences predicted amino acid changes in the sequenced regions, suggesting that the amino acid substitutions reported above are responsible for resistance to ALS-inhibiting herbicides in the respective biotypes. © 1999 Society of Chemical Industry     

Insecticide resistance status of Aedes aegypti (L.) from Colombia

BACKGROUND: To evaluate the insecticide susceptibility status of Aedes aegypti (L.) in Colombia, and as part of the National Network of Insecticide Resistance Surveillance, 12 mosquito populations were assessed for resistance to pyrethroids, organophosphates and DDT. Bioassays were performed using WHO and CDC methodologies. The underlying resistance mechanisms were investigated through biochemical assays and RT‐PCR. RESULTS: All mosquito populations were susceptible to malathion, deltamethrin and cyfluthrin, and highly resistant to DDT and etofenprox. Resistance to lambda‐cyhalothrin, permethrin and fenitrothion ranged from moderate to high in some populations from Chocó and Putumayo states. In Antioquia state, the Santa Fe population was resistant to fenitrothion. Biochemical assays showed high levels of both cytochrome P450 monooxygenases (CYP) and non‐specific esterases (NSE) in some of the fenitrothion‐ and pyrethroid‐resistant populations. All populations showed high levels of glutathione‐S‐transferase (GST) activity. GSTe2 gene was found overexpressed in DDT‐resistant populations compared with Rockefeller susceptible strain. CONCLUSIONS: Differences in insecticide resistance status were observed between insecticides and localities. Although the biochemical assay results suggest that CYP and NSE could play an important role in the pyrethroid and fenitrothion resistance detected, other mechanisms remain to be investigated, including knockdown resistance. Resistance to DDT was high in all populations, and GST activity is probably the main enzymatic mechanism associated with this resistance. The results of this study provide baseline data on insecticide resistance in Colombian A. aegypti populations, and will allow comparison of changes in susceptibility status in this vector over time. Copyright © 2011 Society of Chemical Industry