Effect of diclofop on the membrane potentials of herbicide-resistant and -susceptible annual ryegrass root tips

Electrophysiological measurements were made on root tip cells in the elongation zone of diclofop-methyl-resistant (SR4/84) and -susceptible (SRS2) biotypes of annual ryegrass (Lolium rigidum Gaud.) from Australia. The phytotoxic action of diclofop-methyl (methyl 2-[4-(2′,4′-dichlorophenoxy)phenoxy]propanoate) on susceptible whole plants was completely reversed by a simultaneous application of 2,4-dichlorophenoxyacetic acid (dimethylamine salt). The phytotoxic acid metabolite, diclofop (50 micromolar), depolarized membrane potentials of both biotypes to a steady-state level within 10 to 15 minutes. Repolarization of the membrane potential occurred only in the resistant biotype following removal of diclofop. The resistant biotype has an intrinsic ability to reestablish the electrogenic membrane potential, whereas the susceptible biotype required an exogeneous source of IAA to induce partial repolarization. Both biotypes were susceptible to depolarization by carbonylcyanide-m-chlorophenylhy-drazone (CCCP), and their membrane potentials recovered upon removal of CCCP. A 15-minute pretreatment with p-chloromercuribenzenesulphonic acid (PCMBS) blocked the depolarizing action of diclofop in both biotypes. However, PCMBS had no effect on the activity of CCCP. The action of diclofop appears to involve a site-specific interaction at the plasmalemma in both Lolium biotypes to cause the increased influx of protons into sensitive cells. The differential response of membrane depolarization and repolarization to diclofop treatment may be a significant initial reaction in the eventual phytotoxic action of the herbicide.     

Cross-Resistance to Herbicides in Annual Ryegrass (Lolium rigidum) : III. On the Mechanism of Resistance to Diclofop-Methyl

Annual ryegrass (Lolium rigidum) biotype SLR 31 is resistant to the postemergent graminicide methyl-2-[4-(2,4-dichlorophenoxy)phenoxy]-propanoate (diclofop-methyl). Uptake of [¹⁴C](U-phenyl)diclofop-methyl and root/shoot distribution of radioactivity in susceptible and resistant plants were similar. In both biotypes, diclofop-methyl was rapidly demethylated to the biocidal metabolite diclofop acid which, in turn, was metabolized to ester and aryl-O-sugar conjugates. Susceptible plants accumulated 5 to 15% more radioactivity in dicloflop acid than did resistant plants. Resistant plants had a slightly greater capacity to form nonbiocidal sugar conjugates. Despite these differences, resistant plants retained 20% of ¹⁴C in the biocidal metabolite diclofop acid 192 hours after treatment, whereas susceptible plants, which were close to death, retained 30% in diclofop acid. The small differences in the pool sizes of the active and inactive metabolites are by themselves unlikely to account for a 30-fold difference in sensitivity to the herbicide at the whole plant level. Similar high-pressure liquid chromatography elution patterns of conjugates from both susceptible and resistant biotypes indicated that the mechanisms and the products of catabolism in the biotypes are similar. It is suggested that metabolism of diclofop-methyl by the resistant biotype does not alone explain resistance observed at the whole-plant level. Diclofop acid reduced the electrochemical potential of membranes in etiolated coleoptiles of both biotypes; 50% depolarization required 1 to 4 μm diclofop acid. After removal of diclofop acid, membranes from the resistant biotype recovered polarity, whereas membranes from the susceptible biotype did not. Internal concentrations of diclofop acid 4 h after exposing plants to herbicide were estimated to be 36 to 39 micromolar in a membrane fraction and 16 to 17 micromolar in a soluble fraction. Such concentrations should be sufficient to fully depolarize membranes. It is postulated that differences in the ability of membranes to recover from depolarization are correlated with the resistance response of biotype SLR 31.     

Effects of diclofop and diclofop-methyl on membrane potentials in roots of intact oat, maize, and pea seedlings

Growth and electrophysiological studies in roots of intact diclofop-methyl susceptible and resistant seedlings were conducted to test the hypothesis that the herbicide acts primarily as a proton ionophore. The ester formulation of diclofop, at 0.2 micromolar, completely inhibited root growth in herbicide-susceptible oat (Avena sativa L.) after a 96 hour treatment, but induced only a delayed transient depolarization of the membrane potential in oat root cortical cells. Root growth in susceptible maize (Zea mays L.) seedlings was dramatically reduced by exposure to 0.8 micromolar diclofop-methyl, while the same diclofop-methyl exposure hyperpolarized the membrane potential within 48 hours after treatment. Furthermore, exposure of maize roots to the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP) (50 nanomolar), inhibited growth by only 31%, 96 hours after treatment, while the same CCCP exposure depolarized the resting potential by an average of 32 millivolts. Thus, the protonophore hypothesis cannot account for a differential membrane response to phytotoxic levels of diclofop-methyl in two susceptible species. From the results of others, much of the evidence to support the protonophore hypothesis was obtained using high concentrations of diclofop acid (100 micromolar). At a similar concentration, we also report a rapid (3 minute) diclofop-induced depolarization of the membrane potential in roots of susceptible oat and maize, moderately tolerant barley (Hordeum vulgare L.), and resistant pea (Pisum sativum L.) seedlings. Moreover, 100 micromolar diclofop acid inhibited growth in excised cultured pea roots. In contrast, 100 micromolar diclofop-methyl did not inhibit root growth. Since the membrane response to 100 micromolar diclofop acid does not correspond to differential herbicide sensitivity under field conditions, results obtained with very high levels of diclofop acid are probably physiologically irrelevant. The results of this study suggest that the effect of diclofop-methyl on the membrane potentials of susceptible species is probably unrelated to the primary inhibitory effect of the herbicide on plant growth.     

Cross-Resistance to Herbicides in Annual Ryegrass (Lolium rigidum): IV. Correlation between Membrane Effects and Resistance to Graminicides

The herbicidally active aryloxyphenoxypropionates diclofop acid, haloxyfop acid, and fluazifop acid and the cyclohexanedione sethoxydim depolarized membranes in coleoptiles of eight biotypes of herbicide-susceptible and herbicide-resistant annual ryegrass (Lolium rigidum). Membrane polarity was reduced from −100 millivolts to −30 to −50 millivolts. Membranes repolarized after removal of the compounds only in biotypes with resistance to the compound added. Repolarization was not observed in herbicide-susceptible L. rigidum, nor was it observed in biotypes resistant to triazine, triazole, triazinone, phenylurea, or sulfonylurea herbicides but not resistant to aryloxyphenoxypropionates and cyclohexanediones. Chlorsulfuron, a sulfonylurea herbicide, at a saturating concentration of 1 micromolar, reduced membrane polarity in all biotypes studied by only 15 millivolts. The recovery of membrane potential following the removal of chlorsulfuron was restricted to chlorsulfuron-susceptible and -resistant biotypes that did not exhibit diclofop resistance. These differences in membrane responses are correlated with resistance to dicloflop rather than with resistance to chlorsulfuron. It is suggested that the differences may reflect altered membrane properties of diclofop-resistant biotypes. Further circumstantial evidence for dissimilarity of properties of membranes from diclofop-resistant and diclofop-susceptible ryegrass is provided by observations that K⁺/Na⁺ ratios were significantly higher in coleoptiles from diclofop-resistant biotypes than in coleoptiles from susceptible plants. Intact and excised roots from susceptible biotypes were capable of acidifying the external medium, whereas roots from resistant biotypes were unable to do so. The ineluctable conclusion is that in L. rigidum the phenomena of membrane repolarization and resistance to aryloxyphenoxypropionate and cyclohexanedione herbicides are correlated.     

Purification and Characterization of Acetyl-Coenzyme A Carboxylase from Diclofop-Resistant and -Susceptible Lolium multiflorum

Acetyl-coenzyme A carboxylase (ACCase) was purified >100-fold (specific activity 3.5 units mg-1) from leaf tissue of diclofopresistant and -susceptible biotypes of Lolium multiflorum. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified fractions from both biotypes contained a single 206-kD biotinylated polypeptide. The molecular mass of the native enzyme from both biotypes was approximately 520 kD. In some cases the native dimer from both biotypes dissociated during gel filtration to form a subunit of approximately 224 kD. The inclusion of 5% (w/v) polyethylene glycol 3350 (PEG) in the elution buffer prevented this dissociation. Steady-state substrate kinetics were analyzed in both the presence and absence of 5% PEG. For ACCase from both biotypes, addition of PEG increased the velocity 22% and decreased the apparent Km values for acetyl-coenzyme A (acetyl-CoA), but increased the Km values for bicarbonate and ATP. In the presence of PEG, the Km values for bicarbonate and ATP were approximately 35% higher for the enzyme from the susceptible biotype compared with the resistant enzyme. In the absence of PEG, no differences in apparent Km values were observed for the enzymes from the two biotypes. Inhibition constants (Ki app) were determined for CoA, malonyl-CoA, and diclofop. CoA was an S-hyperbolic (slope replots)-I-hyperbolic (intercept replots) noncompetitive inhibitor with respect to acetyl-CoA, with Ki app values of 711 and 795 [mu]M for enzymes from the resistant and susceptible biotypes, respectively. Malonyl-CoA competitively inhibited both enzymes (versus acetyl-CoA) with Ki app values of 140 and 104 [mu]M for ACCase from resistant and susceptible biotypes, respectively. Diclofop was a linear noncompetitive inhibitor of ACCase from the susceptible biotype and a nonlinear, or S-hyperbolic-I-hyperbolic, noncompetitive inhibitor of ACCase from the resistant biotype. For ACCase from the susceptible biotype the slope (Kis) and intercept (Kii) inhibition constants for diclofop versus acetyl-CoA were 0.08 and 0.44 [mu]M, respectively. ACCase from the resistant biotype had a Ki app value of 6.5 [mu]M. At a subsaturating acetyl-CoA concentration of 50 [mu]M, the Hill coefficients for diclofop binding were 0.61 and 1.2 for ACCase from the resistant and susceptible biotypes, respectively. The Hill coefficients for diclofop binding and the inhibitor replots suggest that the resistant form of ACCase exhibits negative cooperativity in binding diclofop. However, the possibility that the nonlinear inhibition of ACCase activity by diclofop in the enzyme fraction isolated from the resistant biotype is due to the presence of both resistant and susceptible forms of ACCase cannot be excluded.     

Resistant Acetyl-CoA Carboxylase is a Mechanism of Herbicide Resistance in a Biotype of Avena sterilis ssp. ludoviciana

A biotype of Avena sterilis ssp. ludoviciana is highly resistantto a range of herbicides which inhibit a key enzyme in fattyacid synthesis, acetyl-CoA carboxylase (ACCase). Possible mechanismsof herbicide resistance were investigated in this biotype. Acetyl-CoAcarboxylase from the resistant biotype is less sensitive toinhibition by herbicides to which resistance is expressed. I₅₀values for herbicide inhibition of ACCase were 52 to 6 timesgreater in the resistant biotype than in the susceptible biotype.This was the only major difference found between the resistantand susceptible biotypes. The amount of ACCase in the meristemsof the resistant and susceptible is similar during ontogenyand no difference was found in distribution of ACCase betweenthe two biotypes. Uptake, translocation and metabolism of [¹⁴C]diclofop-methylwere not different between the two biotypes. In vivo, ACCaseactivity in the meristems of the susceptible biotype was greatlyinhibited by herbicide application whereas only 25% inhibitionoccurred in the resistant biotype. Depolarisation of plasmamembrane potential by 50 µM diclofop acid was observedin both biotypes and neither biotype showed recovery of themembrane potential following removal of the herbicide. Hence,a modified form of ACCase appears to be the major determinantof resistance in this resistant wild oat biotype. (Received February 10, 1994; Accepted March 11, 1994)     

Physiological tests for early detection of rigid ryegrass (Lolium rigidum Goud.) resistance to fenoxaprop-P

The study aimed to determine the usefulness of isothermal calorimetry and FT-Raman spectroscopy for the early evaluation of rigid ryegrass resistance to fenoxaprop-P ethyl (active ingredient one of aryloxyphenoxypropionate herbicides). The calorimetric measurements were done on the 4-day-old seedlings of susceptible and resistant biotypes of rigid ryegrass (Lolium rigidum Goud.) for 72 h, at 20 °C. It was observed that the specific thermal power–timecurves of the susceptible and resistant biotypes growing on water (control) were qualitatively similar. Herbicides changed the shape of the specific thermal power–time curves of both biotypes. Furthermore, the total specific thermal energy was significantly higher for the seedlings of resistant biotype, growing both on water or herbicide, as compared to the susceptible ones. The analysis of the seedlings’ endosperm, conducted using FT-Raman spectroscopy, showed a weaker intensity of the bands in the spectra derived from the resistant biotype. Differences in the specific thermal power–time curves and FT-Raman spectra between susceptible and resistant biotypes growing on water indicate that the sensitive and resistant biotypes are metabolically and chemically different already in the early stages of the seedling growth. We conclude that isothermal calorimetry and FT-Raman spectroscopy are efficient tools for the early detection of rigid ryegrass resistance to fenoxaprop-P ethyl.     

Cross-Resistance to Herbicides in Annual Ryegrass (Lolium rigidum) : II. Chlorsulfuron Resistance Involves a Wheat-Like Detoxification System

Lolium rigidum Gaud. biotype SLR31 is resistant to the herbicide diclofop-methyl and cross-resistant to several sulfonylurea herbicides. Wheat and the cross-resistant ryegrass exhibit similar patterns of resistance to sulfonylurea herbicides, suggesting that the mechanism of resistance may be similar. Cross-resistant ryegrass is also resistant to the wheat-selective imidazolinone herbicide imazamethabenz. The cross-resistant biotype SLR31 metabolized [phenyl-U-¹⁴C]chlorsulfuron at a faster rate than a biotype which is susceptible to both diclofop-methyl and chlorsulfuron. A third biotype which is resistant to diclofop-methyl but not to chlorsulfuron metabolized chlorsulfuron at the same rate as the susceptible biotype. The increased metabolism of chlorsulfuron observed in the cross-resistant biotype is, therefore, correlated with the patterns of resistance observed in these L. rigidum biotypes. During high performance liquid chromatography analysis the major metabolite of chlorsulfuron in both susceptible and cross-resistant ryegrass coeluted with the major metabolite produced in wheat. The major product is clearly different from the major product in the tolerant dicot species, flax (Linium usitatissimum). The elution pattern of metabolites of chlorsulfuron was the same for both the susceptible and cross-resistant ryegrass but the cross-resistant ryegrass metabolized chlorsulfuron more rapidly. The investigation of the dose response to sulfonylurea herbicides at the whole plant level and the study of the metabolism of chlorsulfuron provide two independent sets of data which both suggest that the resistance to chlorsulfuron in cross-resistant ryegrass biotype SLR31 involves a wheat-like detoxification system.     

Cross-Resistance to Herbicides in Annual Ryegrass (Lolium rigidum): I. Properties of the Herbicide Target Enzymes Acetyl-Coenzyme A Carboxylase and Acetolactate Synthase

Lolium rigidum biotype SR4/84 is resistant to the herbicides diclofop-methyl and chlorsulfuron when grown in the field, in pots, and in hydroponics. Similar extractable activities and affinities for acetyl-coenzyme A of carboxylase (ACCase), an enzyme inhibited by diclofop-methyl, were found for susceptible and resistant L. rigidum. ACCase activity from both biotypes was inhibited by diclofop-methyl, diclofop acid, haloxyfop acid, fluazifop acid, sethoxydim, and tralkoxydim but not by chlorsulfuron or trifluralin. Exposure of plants to diclofop-methyl did not induce any changes in either the extractable activities or the herbicide inhibition kinetics of ACCase. It is concluded that, in contrast to diclofop resistance in L. multiflorum and diclofop tolerance in many dicots, the basis of resistance to diclofop-methyl and to other aryloxyphenoxypropionate and cyclohexanedione herbicides in L. rigidum is not due to the altered inhibition characteristics or expression of the enzyme ACCase. The extractable activities and substrate affinity of acetolactate synthase (ALS), an enzyme inhibited by chlorsulfuron, from susceptible and resistant biotypes of L. rigidum were similar. ALS from susceptible and resistant plants was equally inhibited by chlorsulfuron. Prior exposure of plants to 100 millimolar chlorsulfuron did not affect the inhibition kinetics. It is concluded that resistance to chlorsulfuron is not caused by alterations in either the expression or inhibition characteristics of ALS.     

Effects of Acetyl-Coenzyme A Carboxylase Inhibitors on Root Cell Transmembrane Electric Potentials in Graminicide-Tolerant and -Susceptible Corn (Zea mays L.)

Herbicidal activity of aryloxyphenoxypropionate and cyclohexanedione herbicides (graminicides) has been proposed to involve two mechanisms: inhibition of acetyl-coenzyme A carboxylase (ACCase) and depolarization of cell membrane potential. We examined the effect of aryloxyphenoxypropionates (diclofop and haloxyfop) and cyclohexanediones (sethoxydim and clethodim) on root cortical cell membrane potential of graminicide-susceptible and -tolerant corn (Zea mays L.) lines. The graminicide-tolerant corn line contained a herbicide-insensitive form of ACCase. The effect of the herbicides on membrane potential was similar in both corn lines. At a concentration of 50 [mu]M, the cyclohexanediones had little or no effect on the membrane potential of root cells. At pH 6, 50 [mu]M diclofop, but not haloxyfop, depolarized membrane potential, whereas both herbicides (50 [mu]M) dramatically depolarized membrane potential at pH 5. Repolarization of membrane potential after removal of haloxyfop and diclofop from the treatment solution was incomplete at pH 5. However, at pH 6 nearly complete repolarization of membrane potential occurred after removal of diclofop. In graminicide-susceptible corn, root growth was significantly inhibited by a 24-h exposure to 1 [mu]M haloxyfop or sethoxydim, but cell membrane potential was unaffected. In gramincide-tolerant corn, sethoxydim treatment (1 [mu]M, 48 h) had no effect on root growth, whereas haloxyfop (1 [mu]M, 48 h) inhibited root growth by 78%. However, membrane potential was the same in roots treated with 1 [mu]M haloxyfop or sethoxydim. The results of this study indicate that graminicide tolerance in the corn line used in this investigation is not related to an altered response at the cell membrane level as has been demonstrated with other resistant species.     

Evidence for vacuolar sequestration of paraquat in roots of a paraquat-resistant Hordeum glaucum biotype

Paraquat resistance in the grass weed Hordeum glaucum Steud. has been proposed to result from herbicide sequestration away from the growing points. In the present study, we used roots as a model system to investigate cellular transport of paraquat in resistant (R) and susceptible (S) H. glaucum biotypes. Both time- and concentration-dependent kinetics of paraquat influx across the root cell plasma membrane were similar in the S and R biotype. However, compartmentation analysis indicated greater herbicide accumulation in root vacuoles of the R seedlings. In contrast, the amount of paraquat accumulated in the cytoplasm of S was double that found in R biotype. While paraquat efflux from the cytoplasm back into the external solution was similar in the two biotypes, efflux across the tonoplast from the vacuole back into the cytoplasm was 5 times slower in the R than in the S biotype. At the end of a 48-h efflux period, nearly 7-fold more herbicide was retained in the roots of the R compared with those of the S biotype. These results suggest that paraquat resistance in H. glaucum may be due to the herbicide sequestration in the vacuole.     

Occurrence of a herbicide-resistant acetyl-coenzyme A carboxylase mutant in annual ryegrass (Lolium rigidum) selected by sethoxydim

The spectrum of herbicide resistance was determined in an annual ryegrass (Lolium rigidum Gaud.) biotype (SLR 3) that had been exposed to the grass herbicide sethoxydim, an inhibitor of the plastidic enzyme acetylcoenzyme A carboxylase (ACCase, EC 6.4.1.2), for three consecutive years. This biotype has an 18-fold resistance to sethoxydim and enhanced resistance to other cyclohexanedione herbicides compared with a susceptible biotype (VLR 1). The resistant biotype also has a 47- to >300-fold cross-resistance to the aryloxyphenoxypropanoate herbicides which share ACCase as a target site. No resistance is evident to herbicide with a target site different from ACCase. The absorption of [4-¹⁴C]sethoxydim, the rate of metabolic degradation and the nature of the herbicide metabolites are similar in the resistant and susceptible biotypes. While the total activity of the herbicide target enzyme ACCase is similar in extracts from the two biotypes, the kinetics of herbicide inhibition differ. The concentrations of sethoxydim and tralkoxydim required to inhibit the activity of ACCase by 50% are 7.8 and >9.5 times higher, respectively, in the resistant biotype. The activity of ACCase from the resistant biotype was also less sensitive to aryloxyphenoxypropanode herbicides than the susceptible biotype. The spectrum of resistance at the whole-plant level is correlated with resistance at the ACCase level and confirms that a less sensitive form of the target enzyme endows resistance in biotype SLR 3.Abbreviations ACCase acetyl-coenzyme A carboxylase - AOPP aryloxyphenoxypropanoate - CHD cyclohexanedione - GR₅₀ dose giving 50% reduction of growth - IG₅₀ dose giving 50% reduction of germination - LD₅₀ lethal dose 50 This work was partially supported by The Grains Research and Development Corporation of Australia through a grant to Dr. R. Knight, Department of Plant Science, Waite Agricultural Research Institute. The encouragement and generous support of Dr. R. Knight is gratefully acknowledged.     

Investigations of mechanisms of resistance to bipyridyl herbicides in Arctotheca calendula (L.) Levyns

The mechanism of resistance to diquat and paraquat was investigated in a bipyridyl-herbicide-resistant biotype of Arctotheca calendula (L.) Levyns. No differences were observed in the interactions of these herbicides with Photo-system I, the active site, in thylakoids isolated from resistant and susceptible biotypes. Likewise, absorption of herbicide through the cuticle and gross translocation were identical in plants of the two biotypes. Foliar application of either 25 g ha⁻¹ diquat or 200 g ha⁻¹ paraquat rapidly inhibited CO₂-dependent O₂ evolution of leaf segments of the susceptible biotype. O₂ evolution of leaf segments of the resistant biotype was less affected by these treatments. Fluorescence imaging was used to observe visually, as fluorescence quenching, the penetration of herbicide to the active site. These experiments demonstrated that diquat appears at the active site more slowly in the resistant biotype compared to the susceptible biotype. HCO₃-dependent O₂ evolution of thin leaf slices was less inhibited by diquat in the resistant biotype than in the susceptible biotype. The mechanism of resistance to the bipyridyl herbicides in this biotype of A. calendula is not a result of changes at the active site, decreased herbicide absorption or decreased translocation, but appears to be due to reduced herbicide penetration to the active site.     

On the Mechanism of Resistance to Paraquat in Hordeum glaucum and H. leporinum: Delayed Inhibition of Photosynthetic O(2) Evolution after Paraquat Application

The mechanism of resistance to paraquat was investigated in biotypes of Hordeum glaucum Steud. and H. leporinum Link. with high levels of resistance. Inhibition of photosynthetic O₂ evolution after herbicide application was used to monitor the presence of paraquat at the active site. Inhibition of photosynthetic O₂ evolution after paraquat application was delayed in both resistant biotypes compared with the susceptible biotypes; however, this differential was more pronounced in the case of H. glaucum than in H. leporinum. Similar results could be obtained with the related herbicide diquat. Examination of the concentration dependence of paraquat-induced inhibition of O₂ evolution showed that the resistant H. glaucum biotype was less affected by herbicide compared with the susceptible biotype 3 h after treatment at most rates. The resistant H. leporinum biotype, in contrast, was as inhibited as the susceptible biotype except at the higher rates. In all cases photosynthetic O₂ evolution was dramatically inhibited 24 h after treatment. Measurement of the amount of paraquat transported to the young tissue of these plants 24 h after treatment showed 57% and 53% reductions in the amount of herbicide transported in the case of the resistant H. glaucum and H. leporinum biotypes, respectively, compared with the susceptible biotypes. This was associated with 62% and 66% decreases in photosynthetic O₂ evolution of young leaves in the susceptible H. glaucum and H. leporinum biotypes, respectively, a 39% decrease in activity for the resistant H. leporinum biotype, but no change in the resistant H. glaucum biotype. Photosynthetic O₂ evolution of leaf slices from resistant H. glaucum was not as inhibited by paraquat compared with the susceptible biotype; however, those of resistant and susceptible biotypes of H. leporinum were equally inhibited by paraquat. Paraquat resistance in these two biotypes appears to be a consequence of reduced movement of the herbicide in the resistant plants; however, the mechanism involved is not the same in H. glaucum as in H. leporinum.     

Differential Light Responses of Photosynthesis by Triazine-resistant and Triazine-susceptible Senecio vulgaris Biotypes

Studies were conducted to determine a physiological basis for competitive differences between Senecio vulgaris L. biotypes which are either resistant or susceptible to triazine herbicides. Net carbon fixation of intact leaves of mature plants was higher at all light intensities in the susceptible biotype than in the resistant biotype. Quantum yields measured under identical conditions for each biotype were 20% lower in the resistant than in the susceptible biotype. Oxygen evolution in continuous light measured in stroma-free chloroplasts was also higher at all light intensities in the susceptible biotype than in the resistant biotype. Oxygen evolution in response to flashing light was measured in stroma-free chloroplasts of both biotypes. The steady-state yield per flash of resistant chloroplasts was less than 20% that of susceptible chloroplasts. Susceptible chloroplasts displayed oscillations in oxygen yield per flash typically observed in normal chloroplasts, whereas the pattern of oscillations in resistant chloroplasts was noticeably damped. It is suggested that modification of the herbicide binding site which confers s-triazine resistance may also affect the oxidizing side of photosystem II, making photochemical electron transport much less efficient. This alteration has resulted in a lowered capacity for net carbon fixation and lower quantum yields in whole plants of the resistant type.     

A mechanism of chlorotoluron resistance in Lolium rigidum

Many biotypes of Lolium rigidum Gaud, (annual ryegrass) have developed resistance to herbicides; however, few have developed resistance to phenylurea herbicides. Two biotypes with different histories of herbicide selection pressure were six to eight times less sensitive to the phenylurea herbicide, chlorotoluron, than a susceptible biotype. Resistance was not due to differences in the herbicide target site as oxygen evolution by thylakoids isolated from resistant and susceptible biotypes was similarly inhibited by diuron and chlorotoluron. There was no difference in the uptake and distribution of chlorotoluron into resistant and susceptible plants. There was a twofold greater rate of chlorotoluron detoxification in resistant plants with N-demethylation being a major detoxification reaction. Resistant plants treated with a 3-h pulse of 120 M chlorotoluron recovered net carbon fixation after 42 h, half the time taken by susceptible plants. The mixed-function oxidase inhibitor 1-aminobenzotriazole (70 M) intensified the effects of chlorotoluron in resistant plants when applied in combination with the herbicide for 7 d. 1-Aminobenzotriazole also inhibited the metabolism of chlorotoluron in both resistant and susceptible plants. The cytochrome P-₄₅₀ inhibitor, piperonyl butoxide piperonyl butoxide, interacted with chlorotoluron when applied to plants growing in soil. Chlorotoluron applied with reduced plant dry weight to a greater extent than chlorotoluron alone. It appears, therefore, that enhanced detoxification is the major mechanism of resistance to chlorotoluron in the resistant biotypes studied.Abbreviations ABT 1-aminobenzotriazole - VLR1 Victorian L. rigidum biotype 1 — herbicide susceptible - VLR69 Victorian L. rigidum biotype 69 — herbicide resistant - WLR2 Western Australian L. rigidum biotype 2 — herbicide resistant M.W.M.B, was supported by an Australian Postgraduate Research Award and a supplementary scholarship from the Grains Research and Development Corporation. We are very grateful to Dr. E. Ebert, Ciba Geigy, Basal, Switzerland for providing [¹⁴C]chlorotoluron and standards of chlorotoluron metabolites. We express our gratitude to Dr. John Huppatz of the CSIRO Division of Plant Industry for providing ABT. We also thank Ciba Geigy Australia for providing technical-grade chlorotoluron and formulated phenylurea herbicides.     

Enhanced rates of herbicide metabolism in low herbicide‐dose selected resistant Lolium rigidum

Lolium rigidum is an obligately cross‐pollinated, genetically diverse species and an economically important herbicide resistance‐prone weed. Our previous work has demonstrated that recurrent selection of initially susceptible L. rigidum populations with low herbicide rates results in rapid herbicide resistance evolution. Here we report on the mechanisms endowing low‐dose‐selected diclofop‐methyl resistance in L. rigidum. Results showed that resistance was not due to target‐site ACCase mutations or overproduction, or differential herbicide leaf uptake and translocation. The in vivo de‐esterification of diclofop‐methyl into phytotoxic diclofop acid was rapid and similar in resistant versus susceptible populations. However, further metabolism of diclofop acid into non‐toxic metabolites was always faster in resistant plants than susceptible plants, resulting in up to 2.6‐fold lower level of diclofop acid in resistant plants. This corresponded well with up to twofold higher level of diclofop acid metabolites in resistant plants. The major polar metabolites of diclofop acid chromatographically resembled those of wheat, a naturally tolerant species. Clearly, recurrent selection at reduced herbicide rates selected for non‐target‐site‐based enhanced rates of herbicide metabolism, likely involving cytochrome P450 monooxygenases.     

Germination ecology of Ambrosia artemisiifolia L. and Ambrosia trifida L. biotypes suspected of glyphosate resistance

The germination ecology of Ambrosia artemisiifolia and A. trifida glyphosate susceptible biotypes sampled in marginal areas, was compared with that of the same species but different biotypes suspected of glyphosate resistance, common and giant ragweed, respectively. The suspected resistant biotypes were sampled in Roundup Ready® soybean fields. Within each weed species, the seeds of the biotype sampled in marginal area were significantly bigger and heavier than those of the biotype sampled in the soybean fields. A. artemisiifolia biotypes exhibited a similar dormancy and germination, while differences between A. trifida biotypes were observed. A. artemisiifolia biotypes showed similar threshold temperature for germination, whereas, the threshold temperature of the susceptible A. trifida biotype was half as compared to that of the resistant A. trifida biotype. No significant differences in emergence as a function of sowing depth were observed between susceptible A. artemisiifolia and suspected resistant A. trifida biotype, while at a six-cm seedling depth the emergence of the A. artemisiifolia susceptible biotype was 2.5 times higher than that of the A. trifida suspected resistant biotype. This study identified important differences in seed germination between herbicide resistant and susceptible biotypes and relates this information to the ecology of species adapted to Roundup Ready® fields. Information obtained in this study supports sustainable management strategies, with continued use of glyphosate as a possibility.     

Double herbicide-resistant biotypes of wild oat (<Emphasis Type="Italic">Avena fatua</Emphasis>) display characteristic metabolic fingerprints before and after applying ACCase- and ALS-inhibitors

Plant herbicides inhibit specific enzymes of biosynthetic metabolism, such as acetyl-coenzyme A carboxylase (ACCase) and acetolactate synthase (ALS). Herbicide resistance can be caused by point mutations at the binding domains, catalytic sites and other regions within multimeric enzymes. Direct-injection electrospray mass spectrometry was used for high-throughput metabolic fingerprinting for finding significant differences among biotypes in response to herbicide application. A Mexican biotype of wild oat (Avena fatua) that displays multiple resistances to ACCase- and ALS-inhibiting herbicides was characterized. The dose–response test showed that the double-resistant biotype had a resistance index of 3.58 for pinoxaden and 3.53 for mesosulfuron-methyl. Resistance was accompanied by characteristic mutations at the site of action: an I-1781-L substitution occurred in the ACCase enzyme and an S-653-N mutation was identified within the ALS enzyme. Other mutations were also detected in the genes of the Mexican biotypes. The ionomic fingerprint showed that the multiple-resistant biotype had a markedly different metabolic pattern under control conditions and that this difference was accentuated after herbicide treatment. This demonstrates that single changes of amino acid sequences can produce several holistic modifications in the metabolism of resistant plants compared to susceptible plants. We conclude that in addition to genetic resistance, additional mechanisms of metabolic adaptation and detoxification can occur in multiple-resistant weed plants.     

Resistance to Acetolactate Synthase-Inhibiting Herbicides in Annual Ryegrass (Lolium rigidum) Involves at Least Two Mechanisms

WLR1, a biotype of Lolium rigidum Gaud. that had been treated with the sulfonylurea herbicide chlorsulfuron in 7 consecutive years, was found to be resistant to both the wheat-selective and the nonselective sulfonylurea and imidazolinone herbicides. Biotype SLR31, which became cross-resistant to chlorsulfuron following treatment with the aryloxyphenoxypropionate herbicide diclofop-methyl, was resistant to the wheat-selective, but not the nonselective, sulfonylurea and imidazolinone herbicides. The concentrations of herbicide required to reduce in vitro acetolactate synthase (ALs) activity 50% with respect to control assays minus herbicide for biotype WLR1 was greater than those for susceptible biotype VLR1 by a factor of >30, >30, 7,4, and 2 for the herbicides chlorsulfuron, sulfometuron-methyl, imazapyr, imazathapyr, and imazamethabenz, respectively. ALS activity from biotype SLR31 responded in a similar manner to that of the susceptible biotype VLR1. The resistant biotypes metabolized chlorsulfuron more rapidly than the susceptible biotype. Metabolism of 50% of [phenyl-U-¹⁴C]chlorsulfuron in the culms of two-leaf seedlings required 3.7 h in biotype SLR31, 5.1 h in biotype WLR1, and 7.1 h in biotype VLR1. In all biotypes the metabolism of chlorsulfuron in the culms was more rapid than that in the leaf lamina. Resistance to ALS inhibitors in L. rigidum may involve at least two mechanisms, increased metabolism of the herbicide and/or a herbicide-insensitive ALS.