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)     

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.     

Movement of paraquat in resistant and susceptible biotypes of Erigeron philadelphicus and E. canadensis

Paraquat (1,1'-dimethyl-4,4'-bipyridinium) resistant biotypes of Erigeron philadelphicus and E. canadensis , from fields where paraquat had been used for weed control, showed more than 100 times higher resistance than the susceptible biotype of both plants. Excised leaves of the susceptible biotypes wilted when supplied with more than 5 μ M paraquatat at the cut ends, but those from the resistant biotypes did not wilt even at 500 μ M. Autoradiographs indicated that (¹⁴CH₃)-paraquat taken up through the cut ends was rapidly distributed through the vascular system in leaves of the susceptible biotype, but was barely translocated in leaves of the resistant biotype. The amount of paraquat taken up during 48 h in the resistant biotype was 0.5% of that in the susceptible biotype in light. This difference in paraquat movement may be correlated with paraquat resistance in Erigeron.     

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.     

Atrazine Resistance in a Velvetleaf (Abutilon theophrasti) Biotype Due to Enhanced Glutathione S-Transferase Activity

We previously reported that a velvetleaf (Abutilon theophrasti Medic) biotype found in Maryland was resistant to atrazine because of an enhanced capacity to detoxify the herbicide via glutathione conjugation (JW Gronwald, Andersen RN, Yee C [1989] Pestic Biochem Physiol 34: 149-163). The biochemical basis for the enhanced atrazine conjugation capacity in this biotype was examined. Glutathione levels and glutathione S-transferase activity were determined in extracts from the atrazine-resistant biotype and an atrazine-susceptible or “wild-type” velvetleaf biotype. In both biotypes, the highest concentration of glutathione (approximately 500 nanomoles per gram fresh weight) was found in leaf tissue. However, no significant differences were found in glutathione levels in roots, stems, or leaves of either biotype. In both biotypes, the highest concentration of glutathione S-transferase activity measured with 1-chloro-2,4-dinitrobenzene or atrazine as substrate was in leaf tissue. Glutathione S-transferase measured with 1-chloro-2,4-dinitrobenzene as substrate was 40 and 25% greater in leaf and stem tissue, respectively, of the susceptible biotype compared to the resistant biotype. In contrast, glutathione S-transferase activity measured with atrazine as substrate was 4.4- and 3.6-fold greater in leaf and stem tissue, respectively, of the resistant biotype. Kinetic analyses of glutathione S-transferase activity in leaf extracts from the resistant and susceptible biotypes were performed with the substrates glutathione, 1-chloro-2,4-dinitrobenzene, and atrazine. There was little or no change in apparent K_m values for glutathione, atrazine, or 1-chloro-2,4-dinitrobenzene. However, the V_max for glutathione and atrazine were approximately 3-fold higher in the resistant biotype than in the susceptible biotype. In contrast, the V_max for 1-chloro-2,4-dinitrobenzene was 30% lower in the resistant biotype. Leaf glutathione S-transferase isozymes that exhibit activity with atrazine and 1-chloro-2,4-dinitrobenzene were separated by fast protein liquid (anion-exchange) chromatography. The susceptible biotype had three peaks exhibiting activity with atrazine and the resistant biotype had two. The two peaks of glutathione S-transferase activity with atrazine from the resistant biotype coeluted with two of the peaks from the susceptible biotype, but peak height was three- to fourfold greater in the resistant biotype. In both biotypes, two of the peaks that exhibit glutathione S-transferase activity with atrazine also exhibited activity with 1-chloro-2,4-dinitrobenzene, with the peak height being greater in the susceptible biotype. The results indicate that atrazine resistance in the velvetleaf biotype from Maryland is due to enhanced glutathione S-transferase activity for atrazine in leaf and stem tissue which results in an enhanced capacity to detoxify the herbicide via glutathione conjugation.     

Detoxification of chlorotoluron in a chlorotoluron-resistant biotype of Alopecurus myosuroides. Comparison between cell cultures and whole plants

The metabolism of chlorotoluron in whole plants and cell suspensions was investigated in a previously characterized chlorotoluron-resistant biotype of Alopecurus myosuroides Huds. Both resistant plants and cell suspensions showed a greater capability to metabolize chlorotoluron to non-phytotoxic compounds than the respective susceptible counterparts. Data revealed that although both biotypes degraded chlorotoluron by N -dealkylation and ring-methyl hydroxylation, the resistant biotype showed an enhanced capacity to hydroxylate the parent herbicide. The cytochrome (Cyt) P450 inhibitor 1-aminobenzotriazole (ABT) inhibited the metabolism of chlorotoluron in both resistant and susceptible plants by reducing the formation of non-toxic aryl-hydroxylated derivatives and polar conjugates. N -demethylations were less susceptible to ABT than the other oxidative reactions, but this does not necessarily imply that the second detoxification activity is not Cyt P450, as some P450 activities are more susceptible to ABT than others. Ring-methyl hydroxylation inhibition affected the ability of resistant plants to recover photosynthetic activity after incubation in chlorotoluron, showing a similar fluorescence pattern to susceptible plants in the same conditions without ABT. Fluorescence and metabolism data strongly support the thesis of Cyt P450-mediated 4-methylphenyl hydroxylation as the main route of detoxification of chlorotoluron in the resistant biotype.     

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.     

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.     

Germination and emergence characteristics of triazine-susceptible and triazine-resistant biotypes of Solanum nigrum

1. Seedling emergence patterns of triazine-susceptible and triazine-resistant Solanum nigrum in the field were studied in Wageningen, the Netherlands. Emergence patterns were similar in the first year, but in the second year resistant seedlings emerged faster and the number of resistant seedlings was higher. To explain emergence patterns, a germination experiment was carried out.
2. Seeds from two populations with triazine-susceptible and -resistant biotypes were buried in late autumn and exhumed monthly during spring. Germination was assessed in incubators at different constant temperatures.
3. The lowest temperatures for germination of seeds from the Achterberg population ranged from 20°C on 1 February to 10°C on 1 May for the susceptible biotype, and from 15°C on 1 February to 10°C on 1 May for the resistant biotype. The lowest temperatures for germination of seeds from the Zelhem population ranged from 25°C on 1 February to 10°C on 1 May for the susceptible biotype, and from 15°C on 1 February to 10°C on 1 May for the resistant biotype. The minimum germination temperature of seeds from the resistant biotype appeared to be lower than that of the susceptible biotype.
4. Emergence patterns in the field could be explained by soil temperature and different minimum germination temperature requirements of seeds from the triazine-susceptible and -resistant biotype. This knowledge can be used to manage triazine-resistant biotypes of S. nigrum by the timing of soil cultivation.     

Inheritance and mechanism of resistance to herbicides inhibiting acetolactate synthase in Sonchus oleraceus L.

A biotype of Sonchus oleraceus L. (Compositae) has developed resistance to herbicides inhibiting acetolactate synthase (ALS) following field selection with chlorsulfuron for 8 consecutive years. The aim of this study was to determine the inheritance and mechanism of resistance in this biotype. Determination of ALS activity and inhibition kinetics revealed that K_m and V_max did not vary greatly between the resistant and susceptible biotypes. ALS extracted from the resistant biotype was resistant to five ALS-inhibiting herbicides in an in vitro assay. ALS activity from the resistant biotype was 14 19, 2, 3 and 3 times more resistant to inhibition by chlorsulfuron, sulfometuron, imazethapyr, imazapyr and flumetsulam, respectively, than the susceptible biotype. Hybrids between the resistant and a susceptible biotype were produced, and inheritance was followed through the F₁, F₂ and F₃ generations. F₁ hybrids displayed a uniform intermediate level of resistance between resistant and susceptible parents. Three distinct phenotypes, resistant, intermediate and susceptible, were identified in the F₂ generation following chlorsulfuron application. A segregation ratio of 121 was observed, indicative of the action of a single, nuclear, incompletely dominant gene. F₃ families, derived from intermediate F₂ individuals, segregated in a similar manner. Resistance to herbicides inhibiting ALS in this biotype of S. oleraceus is due to the effect of a single gene coding for a resistant form of the target enzyme, ALS.     

Kinetic Analysis of Resistance to Paraquat in Conyza: Evidence that Paraquat Transiently Inhibits Leaf Chloroplast Reactions in Resistant Plants

Paraquat resistance has been claimed to be due to a sequestration of the herbicide before it reaches chloroplasts. This is based on the sensitivity of photosystem I in isolated thylakoids to paraquat, and autoradiographic analyses showing label from paraquat near veins 4 hours after treatment of a resistant biotype. Conversely, the enzymes of the superoxide detoxification pathway were found to be at constitutively elevated levels in intact class A chloroplasts of the resistant biotype of Conyza bonariensis (L.) Cronq. Evidence is presented here that physiologically active levels of paraquat rapidly inhibit chloroplast function in both the resistant and sensitive biotype, before the first sequestration was visualized. This inhibition is transient (completed in 2 hours) in the resistant biotype and irreversible in the sensitive type. Intact class A chloroplasts of the resistant biotype with or without paraquat are less susceptible to photoinduced membrane damage than the sensitive biotype without paraquat, as measured by ethane evolution. These data support a hypothesis that the ability to prevent superoxide damage keeps the resistant biotype viable while paraquat or its metabolites are being sequestered.     

Phenotypic mechanisms of host resistance against greenbug (Homoptera: Aphididae) revealed by near isogenic lines of wheat

Interactions between biotype E greenbug, Schizaphis graminum (Rondani), and wheat, Triticum aestivum L., were investigated using resistant and susceptible near isogenic lines of the greenbug resistance gene Gb3. In an antixenosis test, the greenbugs preferred susceptible plants to resistant ones when free choice of hosts was allowed. Aphid feeding resulted in quick and severe damage to susceptible plants, which seemed to follow a general pattern spatially and was affected by the position where the greenbugs were initially placed. Symptom of damage in resistant plants resembled senescence. Within-plant distribution of aphids after infestation was clearly different between the two genotypes. Significantly more greenbugs fed on the first (oldest) leaf than on the stem in resistant plants, but this preference was reversed in the susceptible one. After reaching its peak, aphid population on the susceptible plants dropped quickly. All susceptible plants were dead in 10-14 d after infestation due to greenbug feeding. Aphid population dynamics on resistant plants exhibited a multipeak curve. After the first peak, the greenbug population declined slowly. More than 70% of resistant plants were killed 47 d after infestation. Performance of both biotype E and I greenbugs on several Gb3-related wheat germplasm lines were also examined. It seems that the preference-on-stem that was characteristic of biotype E greenbugs on the susceptible plants was aphid biotype- and host genotype-dependent. Results from this study suggested that antixenosis, antibiosis, and tolerance in the resistant plants of wheat might all contribute to resistance against greenbug feeding.     

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.     

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.     

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.     

Paraquat resistance in conyza

A biotype of Conyza bonariensis (L.) Cronq. (identical to Conyza linefolia in other publications) originating in Egypt is resistant to the herbicide 1,1′-dimethyl-4,4′-bipyridinium ion (paraquat). Penetration of the cuticle by [¹⁴C]paraquat was greater in the resistant biotype than the susceptible (wild) biotype; therefore, resistance was not due to differences in uptake. The resistant and susceptible biotypes were indistinguishable by measuring in vitro photosystem I partial reactions using paraquat, 6,7-dihydrodipyrido [1,2-α:2′,1′-c] pyrazinediium ion (diquat), or 7,8-dihydro-6H-dipyrido [1,2-α:2′,1′-c] [1,4] diazepinediium ion (triquat) as electron acceptors. Therefore, alteration at the electron acceptor level of photosystem I is not the basis for resistance. Chlorophyll fluorescence measured in vivo was quenched in the susceptible biotype by leaf treatment with the bipyridinium herbicides. Resistance to quenching of in vivo chlorophyll fluorescence was observed in the resistant biotype, indicating that the herbicide was excluded from the chloroplasts. Movement of [¹⁴C] paraquat was restricted in the resistant biotype when excised leaves were supplied [¹⁴C]paraquat through the petiole. We propose that the mechanism of resistance to paraquat is exclusion of paraquat from its site of action in the chloroplast by a rapid sequestration mechanism. No differential binding of paraquat to cell walls isolated from susceptible and resistant biotypes could be detected. The exact site and mechanism of paraquat binding to sequester the herbicide remains to be determined.     

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.     

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.     

Pollen Expression of Herbicide Target Site Resistance Genes in Annual Ryegrass (Lolium rigidum)

Herbicide resistance can occur either through target-site insensitivity or by nontarget site-based mechanisms. Two herbicide-resistant biotypes of Lolium rigidum Gaud., one resistant to acetolactate synthase (ALS)-inhibiting herbicides (biotype WLR1) and the other resistant to acetyl CoA carboxylase (ACCase)-inhibiting herbicides (biotype WLR96) through target-site insensitivity at the whole plant and enzymic levels, were found to express this resistance in the pollen. Pollen produced by resistant biotypes grew uninhibited when challenged with herbicide, whereas that from a susceptible biotype was inhibited. A third biotype, SLR31, resistant to ACCase-inhibiting and certain ALS-inhibiting herbicides at the whole plant level through nontarget site-based mechanisms, did not exhibit this expression in the pollen. The technique described may form the basis for a rapid screen for certain nuclear-encoded, target site-based herbicide-resistance mechanisms.     

The mechanism of resistance to paraquat is strongly temperature dependent in resistant Hordeum leporinum Link and H. glaucum Steud.

Paraquat-resistant biotypes of the closely-related weed species Hordeum leporinum Link and H. glaucum Steud. are highly resistant to paraquat when grown during the normal winter growing season. However, when grown and treated with paraquat in summer, these biotypes are markedly less resistant to paraquat. This reduced resistance to paraquat in summer is primarily a result of increased temperature following herbicide treatment. The mechanism governing this decrease in resistance at high temperature was examined in H. leporinum. No differences were observed between susceptible and resistant biotypes in the interaction of paraquat with isolated thylakoids when assayed at 15, 25, or 35 °C. About 98 and 65% of applied paraquat was absorbed through the leaf cuticle of both biotypes at 15 and 30 °C, respectively. Following application to leaves, more herbicide was translocated in a basipetal direction in the susceptible biotype compared to the resistant biotype at 15 °C. However, at 30 °C more paraquat was translocated in a basipetal direction in the resistant biotype. Photosynthetic activity of young leaf tissue from within the leaf sheath which had not been directly exposed to paraquat was measured 24 h after treatment of plants with para. quat. This activity was inhibited in the susceptible biotype when plants were maintained at either 15 °C or 30 °C after treatment. In contrast, photosynthetic activity of such tissue of the resistant biotype was not inhibited when plants were maintained at 15 °C after treatment, but was inhibited at 30 °C. The mechanism of resistance in this biotype of H. leporinum correlates with decreased translocation of paraquat and decreased penetration to the active site. This mechanism is temperature sensitive and breaks down at higher temperatures.We are grateful to Zeneca Agrochemicals, Jealotts Hill, Berkshire, UK who provided [¹⁴C]paraquat. E.P. was supported through a Ph.D. scholarship from the Australian International Development Assistance Bureau and C.P. was the recipient of an Australian Research Council Postdoctoral Fellowship.