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.     

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     

Mechanisms of Resistance to Aryloxyphenoxypropionate Herbicides in Two Resistant Biotypes ofAlopecurus myosuroides(blackgrass): Herbicide Metabolism as a Cross-Resistance Mechanism

The mechanisms of AOPP herbicide resistance in twoAlopecurus myosuroidesbiotypes were investigated. Resistant biotype Peldon A1, which is highly resistant to the phenyl-urea chlorotoluron, is moderately resistant to the AOPP herbicides diclofop-methyl, fenoxaprop-ethyl, fluazifop-P-butyl, and the CHD tralkoxydim. Resistant biotype Lincs. E1, which is only moderately resistant to chlorotoluron, is highly resistant to the AOPP herbicide fenoxaprop-ethyl, and moderately resistant to diclofop-methyl, fluazifop-P-butyl, and the CHD tralkoxydim. There is no clear evidence of resistance to the CHD sethoxydim in either biotype. Both Peldon A1 and Lincs. E1 exhibited moderately enhanced metabolism of diclofop-methyl. The approximate half life of diclofop was 8 and 9 HAT, respectively, compared to 17 HAT for the susceptible Rothamsted biotype. Peldon A1 showed moderately enhanced metabolism of fenoxaprop-P-ethyl. However, in the highly resistant Lincs. E1, fenoxaprop-P-ethyl metabolism rates were intermediate between Peldon A1 and the susceptible biotype. Fenoxaprop-P-ethyl metabolism inA. myosuroideswas not significantly reduced by inhibitors of cytochrome P450: PBO, tetcyclasis, or ABT. While enhanced herbicide metabolism can account for the moderate AOPP/CHD resistance observed in Peldon A1in vivo, it cannot account in total for fenoxaprop-ethyl resistance in Lincs. E1. Lincs. E1 may possess one or more additional resistance mechanism.     

Resistance of Raphanus raphanistrum to chlorsulfuron in the Republic of South Africa

Herbicide resistance poses a substantial threat to the agricultural industry throughout the world and during the past decade several reports regarding herbicide resistance have been published. Raphanus raphanistrum L., from two wheat farms located in the winter rainfall region of South Africa, showed indications of resistance to chlorsulfuron. Seeds from these suspected resistant biotypes as well as seeds from a susceptible biotype were collected and transported to the ARC-Small Grain Institute for herbicide resistance studies. Herbicides registered for R. raphanistrum control, i.e. chlorsulfuron, MCPA and bromoxynil, were used in this study. Significant differences in the degree of control were found between the susceptible and two resistant biotypes, when treated with chlorsulfuron. The LD₅₀ values for the resistant biotypes (WR 1 & WR 2) were 45 and 11.3 g a.i. ha^–1, respectively, whereas the LD₅₀ value for the susceptible biotype was 5.6 g a.i. ha^–1. The almost eightfold difference between the susceptible and resistant biotype (WR 1), indicated that resistance has developed to chlorsulfuron. Only twofold resistance was established between the other resistant biotype (WR 2) and the susceptible biotype. Significant differences between herbicide rates were also established with the MCPA and bromoxynil experiments. No significant difference could, however, be found between the susceptible and resistant biotypes when treated with MCPA and bromoxynil, indicating the importance of different modes of action of herbicide as a strategy to prevent herbicide resistance.     

麦田抗性生物型荠菜对苯磺隆的抗性机制研究

为明确抗性生物型荠菜对苯磺隆的抗性机制,分别测定了苯磺隆对抗性和敏感生物型荠菜体内乙酰乳酸合成酶(ALS)、谷胱甘肽-S-转移酶(GSTs)、超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)的影响。结果表明:离体条件下,抗性生物型荠菜体内ALS对苯磺隆的敏感性明显降低,苯磺隆对荠菜抗性和敏感生物型ALS的抑制中浓度(I50)分别为0.722 8和0.052 1 μmol/L,抗性与敏感生物型I50的比值为13.87;活体条件下,施用苯磺隆后,抗性和敏感生物型荠菜ALS活性均受到一定程度的抑制,但抗性生物型ALS活性受到抑制后能逐渐恢复,而敏感生物型则不能恢复;经苯磺隆处理后,抗性生物型GSTs相对活力明显高于敏感生物型,而抗性和敏感生物型体内POD、SOD和CAT相对活力无明显差异。研究表明,抗性生物型荠菜体内ALS对苯磺隆敏感性降低是其抗药性产生的原因之一,而GSTs对苯磺隆代谢能力的差异也可能与荠菜对苯磺隆的抗性有关。     

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.     

Mechanism of resistance to bensulfuron-methyl in Alisma plantago-aquatica biotypes from Portuguese rice paddy fields

Two Alisma plantago‐aquatica biotypes resistant to bensulfuron‐methyl were detected in rice paddy fields in Portugal’s Mondego (biotype T) and Tagus and Sorraia (biotype Q) River valleys. The fields had been treated with bensulfuron‐methyl‐based herbicide mixtures for 4–6 years. In order to characterize the resistant (R) biotypes, dose–response experiments, absorption and translocation assays, metabolism studies and acetolactate synthase (ALS) activity assays were performed. There were marked differences between R and susceptible (S) biotypes, with a resistance index (ED₅₀R/S) of 500 and 6.25 for biotypes Q and T respectively. Cross‐resistance to azimsulfuron, cinosulfuron and ethoxysulfuron, but not to metsulfuron‐methyl, imazethapyr, bentazone, propanil and MCPA was demonstrated. No differences in the absorption and translocation of ¹⁴C‐bensulfuron‐methyl were found between the biotypes studied. Maximum absorption attained 1.12, 2.02 and 2.56 nmol g⁻¹ dry weight after 96 h incubation with herbicide, for S, Q and T biotypes respectively. Most of the radioactivity taken up by the roots was translocated to shoots. Bensulfuron‐methyl metabolism in shoots was similar in all biotypes. The R biotypes displayed a higher level of ALS activity than the S biotype, both in the presence and absence of herbicide and the resistance indices (IC₅₀R/S) were 20 197 and 10 for biotypes Q and T respectively. These data confirm for the first time that resistance to bensulfuron‐methyl in A. plantago‐aquatica is target‐site‐based. In practice, to control target site R biotypes, it would be preferable to use mixtures of ALS inhibitors with herbicides with other modes of action.     

Physiological and molecular basis for ALS inhibitor resistance in Bromus tectorum biotypes

Primisulfuron‐resistant (AR and MR) and ‐susceptible (AS and MS) Bromus tectorum biotypes were collected from a Poa pratensis field at Athena, Oregon, and in research plots at Madras, Oregon. Studies were conducted to characterize the resistance of the B. tectorum biotypes. Whole plant bioassay and acetolactate synthase (ALS) enzyme assay revealed that the AR biotype was highly resistant to the sulfonylurea (SU) herbicides, primisulfuron and sulfosulfuron and to a sulfonylaminocarbonyltriazolinone (SCT) herbicide, propoxycarbazone‐sodium. However, the AR biotype was not resistant to imazamox, an imidazolinone (IMI) herbicide. Results of the whole plant bioassay studies showed that the MR biotype was moderately resistant to all ALS inhibitors tested. However, there were no differences in ALS sensitivities between the MR and MS biotypes. The nucleotide and amino acid sequence analysis of the als gene demonstrated a single‐point mutation from C to T, conferring the exchange of the amino acid proline to serine at position 197 in the AR biotype. However, this mutation was not found in the MR biotype. Results of this research indicate that: the resistance of the AR biotype to SU and SCT herbicides is based on an altered target site due to a single‐point mutation; resistance in the MR biotype is not due to a target site mutation.     

Sulfonylurea resistance in Stellaria media [L.] Vill.

A sulfonylurea resistant biotype of common chickweed (Stellaria media L. Vill.) was found in a field treated with chlorsulfuron or metsulfuron for eight consecutive years. In pot experiments the biotype was resistant to postemergence treatments with the following acetolactate synthase (ALS) inhibitors: chlorsulfuron, metsulfuron, tribenuron, triasulfuron, rimsulfuron, sulfometuron, flumetsulam and imazapyr. The level of resistance to chlorsulfuron and sulfometuron was higher than to the other sulfonylurea herbicides. Whereas the level of cross resistance to the triazolopyrimidine herbicide, flumetsulam was comparable to that of metsulfuron, that of imazapyr was significantly lower. In contrast to imazapyr the biotype was not resistant to imazethapyr, an other imidazolinone herbicide. ALS in vitro assays revealed that resistance was due to an ALS enzyme that was less sensitive to ALS inhibiting herbicides. Herbicides with different modes of action were equally effective on the susceptible and resistant biotypes.     

Glyphosate resistance in common ragweed ( A mbrosia artemisiifolia L.) from Mississippi,USA

Glyphosate is one of the most commonly used broad‐spectrum herbicides over the last 40 years. Due to the widespread adoption of glyphosate‐resistant (GR) crop technology, especially corn, cotton and soybean, several weed species have evolved resistance to this herbicide. Research was conducted to confirm and characterize the magnitude and mechanism of glyphosate resistance in two GR common ragweed ( A mbrosia artemisiifolia L.) biotypes from Mississippi, USA. A glyphosate‐susceptible (GS) biotype was included for comparison. The effective glyphosate dose to reduce the growth of the treated plants by 50% for the GR1, GR2 and GS biotypes was 0.58, 0.46 and 0.11 kg ae ha^?1, respectively, indicating that the level of resistance was five and fourfold that of the GS biotype for GR1 and GR2, respectively. Studies using ¹⁴ C‐glyphosate have not indicated any difference in its absorption between the biotypes, but the GR1 and GR2 biotypes translocated more ¹⁴ C‐glyphosate, compared to the GS biotype. This difference in translocation within resistant biotypes is unique. There was no amino acid substitution at codon 106 that was detected by the 5‐enolpyruvylshikimate‐3‐phosphate synthase gene sequence analysis of the resistant and susceptible biotypes. Therefore, the mechanism of resistance to glyphosate in common ragweed biotypes from Mississippi is not related to a target site mutation or reduced absorption and/or translocation of glyphosate.     

Sulfonylurea herbicide resistance in Sonchus asper biotypes in Alberta, Canada

Summary Two Sonchus asper (spiny annual sow-thistle) biotypes, suspected of being resistant to the sulfonylurea herbicide metsulfuron-methyl, were collected in 1996 from two barley ( Hordeum vulgare ) fields in central Alberta, Canada. Both fields had received at least six applications of acetolactate synthase (ALS)-inhibiting herbicide(s). The responses of the two resistant (R) biotypes and two susceptible (S) biotypes to several sulfonylurea herbicides, and to herbicides and herbicide mixtures with other mechanisms of action, were compared. Both R biotypes were highly resistant to all sulfonylurea herbicides, but their control with other herbicides and mixtures was effective and comparable to that of the S biotypes. ALS extracted from an R biotype was about 440 times more resistant to metsulfuron-methyl than that of an S biotype, indicating that resistance was conferred by an ALS enzyme that was less sensitive to inhibition by the herbicide. Competitiveness and seed production of S. asper varied among biotypes, but the differences were probably the result of ecotype differences rather than resistance or susceptibility to sulfonylurea herbicides. This is the first reported occurrence of target site-based S. asper resistance to ALS-inhibiting herbicides.     

麦田抗性生物型猪殃殃对苯磺隆的抗性机制

为探讨猪殃殃Galium aparine抗药性生物型(R)对苯磺隆的抗性机制,测定了苯磺隆对猪殃殃抗性、敏感(S)生物型体内靶标酶 、代谢酶 及抗氧化酶 影响的差异。离体试验结果表明,苯磺隆对R、S生物型猪殃殃ALS的抑制中量(IC50)分别为0.682、0.718 μg/L(有效剂量),R、S生物型猪殃殃ALS对苯磺隆的敏感性不存在差异。活体试验结果表明,苯磺隆茎叶喷雾处理后,R、S生物型ALS活力均表现为先上升,但S生物型上升幅度小,且随后快速下降,第3 d即回落至对照之下,并维持在低于对照的水平,而R生物型ALS活力在第2 d可达对照的4.10倍,第5 d 基本回落至对照水平,之后基本维持在对照水平;R生物型GSTs活力在第1 d即开始上升,最高可达对照的 2.40 倍,而S生物型则表现为先下降,然后小幅回升,最高为对照的1.61倍,两者在10 d左右均回落至对照水平;R生物型SOD活力与对照基本相同,而S生物型虽略有下降,但R、S间不存在显著差异;两者POD活力虽均有大幅提高,但亦不存在显著差异。结果表明,低水平抗药性生物型猪殃殃对苯磺隆产生抗性的原因可能是ALS过量表达及GSTs对苯磺隆的代谢作用加强,而不是由于ALS的敏感性下降,同时POD、SOD在减轻药害中也具有一定作用。     

Differential atrazine interference with the Hill reaction of isolated chloroplasts from Chenopodium album L. biotypes

Atrazine [2-chloro-4-(ethylaminol-6-(isopropyl-amino)-s-triazine] resistant biotypes of lamb's quarters (Chenopodium athum L) were reported in the maize growing areas of Ontario, where the herbicide had been used fur a number of years. Field samples from four locations proved tolerant to higber than recommended rates of atrazine in controlled environment screening trials. A resistant biotype was not killed with up to 40 kg/ha atrazine. Diuron at 5 x10-5 M inhibited the Hill reaction with isolated chloroplasts of resisiant and susceptible biotypes of lamb's-quarters. However, with 10-4 M atrazine, the photochemical activity was inhibited in chloroplasts isolated from the susceptible biotype but not in chloroplasts from the resisiant biotype. With maize chloroplasts, inhibition with 10-4 M atrazine was the same as with the susceptible biotype of lamb's-quarters. These studies suggested that a new mechanism of intraspecific resistance in lamb's quarters to atrazine was involved, other than differences in uptake, translocation and metabolism reported with interspecific comparisons involving the s-triazines and other herbicides, It was also concluded that atrazine and diuron did not have precisely the same mechanism of action as photosynthetic inhibitors with lamb's-quarters, and that external and or internal structure or function of chloroplasts in relation to atrazine inhibition can vary significantly even in biotypes of the same species.     

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.     

The effect of temperature on glutathione S-transferase activity and glutathione content in Alopecurus myosuroides (black grass) biotypes susceptible and resistant to herbicides

Herbicide‐resistant populations of Alopecurus myosuroides (black grass) have become widespread throughout the UK since the early 1980s. Previous observations in this laboratory have demonstrated that natural climatic fluctuations caused increases in endogenous glutathione S‐transferase (GST) enzyme activity in A. myosuroides plants as they mature, which is thought to be linked to herbicide resistance in this species. The present study has investigated the effects of plant growth at 10°C and 25°C, and reports GST specific activity and glutathione (GSH) pool size in resistant and susceptible A. myosuroides biotypes. Findings demonstrate differences in GST activity between resistant and susceptible populations, which are transient at lower growth temperatures. The GSH pool size was elevated at lower growth temperature in both biotypes. We speculate that these endogenous responses are part of a natural mechanism of acclimation to environmental change in this species and suggest that resistant plants are more able to adapt to environmental stress, as indicated in this instance by temperature change. These observations imply that the control of resistant A. myosuroides by graminicides may be more effective when applied at lower temperatures and at earlier growth stages.     

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     

Conyza albida: a new biotype with ALS inhibitor resistance

Summary A biotype of Conyza albida resistant to imazapyr was discovered on a farm in the province of Seville, Spain, on land that had been continuously treated with this herbicide. This is the first reported occurrence of target site resistance to acetolactate synthase (ALS)-inhibiting herbicides in C. albida . In order to characterize this resistant biotype, dose–response experiments, absorption and translocation assays, metabolism studies, ALS activity assays and control with alternative herbicides were performed. Dose–response experiments revealed a marked difference between resistant (R) and susceptible (S) biotypes with a resistance factor [ED₅₀(R)/ED₅₀(S)] of 300. Cross-resistance existed with amidosulfuron, imazethapyr and nicosulfuron. Control of both biotypes using alternative herbicides was good using chlorsulfuron, triasulfuron, diuron, simazine, glyphosate and glufosinate. The rest of the herbicides tested did not provide good control for either biotype. There were no differences in absorption and translocation between the two biotypes, the maximum absorption reached about 15%, and most of the radioactivity taken up remained in the treated leaf. The metabolism pattern was similar and revealed that both biotypes may form polar metabolites with similar retention time (R_f). The effect of several ALS inhibitors on ALS (target site) activity measured in leaf extracts from both biotypes was investigated. Only with imazapyr and imazethapyr did the R biotype show a higher level of resistance than the S biotype [I₅₀ (R)/I₅₀(S) value of 4.0 and 3.7 respectively]. These data suggest that the resistance to imazapyr found in the R biotype of C. albida results primarily from an altered target site.     

Inhibition of photosynthesis in intact plants of biotypes resistant or susceptible to atrazine and cross-resistance to other herbicides

A concentration of atrazine of 0?1 mm 1^?1 in the nutrient solution resulted in complete inhibition of photosynthesis in intact leaves of susceptible biotypes of Amaranthus retroflexus L., Polygonum lapathifolium L., Chenopodium album L., Solanum nigrum L., Poa annua L. and Stellaria media (L.) Vill. within a few hours of treatment, whereas the inhibition of the resistant biotypes by the same concentration varied from small to moderate. In contrast, diuron (10 or 20 μm 1^?1) produced only minor differences between resistant and susceptible biotypes. The influence of some other herbicides on photosynthesis of these resistant biotypes was also smaller than that on the susceptible biotypes. This cross-resistance was evaluated with the resistance factor for intact leaves of Brassica napus L., A. retroflexus, and S. nigrum. This factor is equal to the ratio of the herbicide concentration in leaves of the resistant biotype to that in leaves of the susceptible biotype with inhibition to half-maximum rate of photosynthesis. This concentration in the leaves was calculated from the concentration in the nutrient solution, and the total transpiration divided by leaf area from the beginning of the herbicide treatment until the moment of half-maximum of photosynthesis. The resistance factors for intact leaves of A. retroflexus, S. nigrum and B. napus were 26–30 for atraton, 3–7 for metamitron, 2–9 for bromacil, 3–5 for monolinuron, 1 for diuron and < 1 for bentazone. For isolated chloroplasts much higher values have been reported. The reason for this discrepancy is not clear. A somewhat higher resistance factor (around 50–60) was derived after infiltration of detached leaves of these species with atraton solutions.     

Molecular basis of diverse responses to acetolactate synthase-inhibiting herbicides in sulfonylurea-resistant biotypes of Schoenoplectus juncoides

Sulfonylurea-resistant biotypes of Schoenoplectus juncoides were collected from Nakafurano, Shiwa, Matsuyama, and Yurihonjyo in Japan. All of the four biotypes showed resistance to bensulfuron-methyl and thifensulfuron-methyl in whole-plant experiments. The growth of the Nakafurano, Shiwa, and Matsuyama biotypes was inhibited by imazaquin-ammonium and bispyribac-sodium, whereas the Yurihonjyo biotype grew normally after treatment with these herbicides. The herbicide concentration required to inhibit the acetolactate synthase (ALS) enzyme by 50% (I₅₀), obtained using in vivo ALS assays, indicated that the four biotypes were > 10-fold more resistant to thifensulfuron-methyl than a susceptible biotype. The Nakafurano, Shiwa, and Matsuyama biotypes exhibited no or little resistance to imazaquin-ammonium, whereas the Yurihonjyo biotype exhibited 6700-fold resistance to the herbicide. The Nakafurano and Shiwa biotypes exhibited no resistance to bispyribac-sodium, but the Matsuyama biotype exhibited 21-fold resistance and the Yurihonjyo biotype exhibited 260-fold resistance to the herbicide. Two S. juncoides ALS genes (ALS1 and ALS2) were isolated and each was found to contain one intron and to encode an ALS protein of 645 amino acids. Sequencing of the ALS genes revealed an amino acid substitution at Pro₁₉₇ in either encoded protein (ALS1 or ALS2) in the biotypes from Nakafurano (Pro₁₉₇ → Ser₁₉₇), Shiwa (Pro₁₉₇ → His₁₉₇), and Matsuyama (Pro₁₉₇ → Leu₁₉₇). The ALS2 of the biotype from Yurihonjyo was found to contain a Trp₅₇₄ → Leu₅₇₄ substitution. The relationships between the responses to ALS-inhibiting herbicides and the amino acid substitutions, which are consistent with previous reports in other plants, indicate that the substitutions at Pro₁₉₇ and Trp₅₇₄ are the basis of the resistance to sulfonylureas in these S. juncoides biotypes.     

Glutathione transferases and herbicide detoxification in suspension-cultured cells of giant foxtail (Setaria faberi)

Glutathione transferases (GSTs) catalysing the conjugation of 1-chloro-2,4-dinitrobenzene, the chloro-s-triazine herbicide atrazine, the chloroacetanilide herbicides metolachlor and alachlor and the diphenyl ether herbicide fluorodifen have been identified in suspension-cultured cells derived from the grass weed giant foxtail (Setaria faberi Herrm.). In contrast to suspension-cultured cells of maize, where atrazine-conjugating GSTs are lost during de-differentiation, the GSTs active toward this herbicide in S. faberi plants were also expressed in cultures, suggesting that these isoenzymes are subject to different regulation in the crop and weed. As a result, glutathione conjugation was the major route of atrazine metabolism in S. faberi cultures. Activities of these GSTs were maximal three days after sub-culturing when the cells were dividing most actively, when they were determined to be in the order CDNB>alachlor>metolachlor= fluorodifen>atrazine. This indicated that GSTs which are enhanced during cell division can metabolise herbicides. On the basis of activity per mg protein, GST activities in the cultures were between 20 and 60-fold higher than those determined in the foliage of S. faberi seedlings. The GSTs with activity towards CDNB were resolved into three peaks following anion-exchange chromatography at pH 7·8 using Q-Sepharose. Peak 1 GSTs were not retained, while peak 2 and peak 3 were sequentially resolved with an increasing concentration of salt. Peak 1 GSTs showed activity toward metolachlor and atrazine but showed little activity toward fluorodifen. Peak 2 and peak 3 GSTs were active toward atrazine and metolachlor, with peak 3 being particularly associated with activity toward fluorodifen. The GSTs in these peaks were then further purified using S-hexyl-glutathione-agarose affinity chromatography. In each case, the affinity-bound fraction of the GSTs consisted of 28 kDa and 26 kDa polypeptides, suggesting that the GST isoenzymes in S. faberi cultures are composed of related subunits. Our results demonstrate that the GST isoenzymes involved in herbicide metabolism in suspension cultures of a grass weed show a similar level of complexity to that determined in maize cell cultures. © 1998 SCI