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.     

Inheritance of bipyridyl herbicide resistance in Arctotheca calendula and Hordeum leporinum

The mode of inheritance of resistance to bipyridyl herbicides in bipyridyl-resistant biotypes of Arctotheca calendula and of Hordeum leporinum was investigated. F₁ plants from reciprocal crosses between diquat-resistant and -susceptible plants of A. calendula showed an intermediate response to diquat application that was nuclearly inherited. Treatment of F₂ plants with 100 g ai ha^-1 of diquat or 800 g ai ha^-1 of paraquat killed all homozygous-susceptible plants, caused severe injury to heterozygous plants but only slight or no injury to homozygous-resistant plants. Back crosses of F₁ to susceptible plants exhibited intermediate and susceptible phenotypes. The observed segregation ratios in F₂ and test-cross populations fitted predicted segregation ratios, 1:2:1 (R:I:S) and 1:1 (I:S) respectively, showing that bipyridyl resistance is conferred by a single incompletely-dominant gene. Biotypes of paraquat-resistant and -susceptible H. leporinum were crossed reciprocally. F₁ plants from reciprocal crosses showed an intermediate response to paraquat application. The F₂ progeny showed segregation ratios that fitted the predicted segregation ratio of 1:2:1 (R:I:S) forinheritance of resistance being governed by a single partially-dominant gene.     

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.     

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.     

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.     

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.     

Glyphosate,paraquat and ACCase multiple herbicide resistance evolved in a <Emphasis Type="Italic">Lolium rigidum</Emphasis> biotype

Glyphosate is the world’s most widely used herbicide. A potential substitute for glyphosate in some use patterns is the herbicide paraquat. Following many years of successful use, neither glyphosate nor paraquat could control a biotype of the widespread annual ryegrass (Lolium rigidum), and here the world’s first case of multiple resistance to glyphosate and paraquat is confirmed. Dose–response experiments established that the glyphosate rate causing 50% mortality (LD₅₀) for the resistant (R) biotype is 14 times greater than for the susceptible (S) biotype. Similarly, the paraquat LD₅₀ for the R biotype is 32 times greater than for the S biotype. Thus, based on the LD₅₀ R/S ratio, this R biotype of L. rigidum is 14-fold resistant to glyphosate and 32-fold resistant to paraquat. This R biotype also has evolved resistance to the acetyl-coenzyme A carboxylase (ACCase) inhibiting herbicides. The mechanism of paraquat resistance in this biotype was determined as restricted paraquat translocation. Resistance to ACCase-inhibiting herbicides was determined as due to an insensitive ACCase. Two mechanisms endowing glyphosate resistance were established: firstly, a point mutation in the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene, resulting in an amino acid substitution of proline to alanine at position 106; secondly, reduced glyphosate translocation was found in this R biotype, indicating a co-occurrence of two distinct glyphosate resistance mechanisms within the R population. In total, this R biotype displays at least four co-existing resistance mechanisms, endowing multiple resistance to glyphosate, paraquat and ACCase herbicides. This alarming case in the history of herbicide resistance evolution represents a serious challenge for the sustainable use of the precious agrochemical resources such as glyphosate and paraquat.     

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.     

Mechanism of Sulfonylurea Herbicide Resistance in the Broadleaf Weed, Kochia scoparia

Selection of kochia (Kochia scoparia) biotypes resistant to the sulfonylurea herbicide chlorsulfuron has occurred through the continued use of this herbicide in monoculture cereal-growing areas in the United States. The apparent sulfonylurea resistance observed in kochia was confirmed in greenhouse tests. Fresh and dry weight accumulation in the resistant kochia was 2- to >350-fold higher in the presence of four sulfonylurea herbicides as compared to the susceptible biotype. Acetolactate synthase (ALS) activity isolated from sulfonylurea-resistant kochia was less sensitive to inhibition by three classes of ALS-inhibiting herbicides, sulfonylureas, imidazolinones, and sulfonanilides. The decrease in ALS sensitivity to inhibition (as measured by the ratio of resistant I₅₀ to susceptible I₅₀) was 5- to 28-fold, 2- to 6-fold, and 20-fold for sulfonylurea herbicides, imidazolinone herbicides, and a sulfonanilide herbicide, respectively. No differences were observed in the ALS-specific activities or the rates of [¹⁴C]chlorsulfuron uptake, translocation, and metabolism between susceptible and resistant kochia biotypes. The K_m values for pyruvate using ALS from susceptible and resistant kochia were 2.13 and 1.74 mm, respectively. Based on these results, the mechanism of sulfonylurea resistance in this kochia biotype is due solely to a less sulfonylurea-sensitive ALS enzyme.     

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.     

Growth and Photosynthetic Characteristics of Two Biotypes of the Weed Black-grass (Alopecurus myosuroides Huds.) Resistant and Susceptible to the Herbicide Chlorotoluron

Response of two biotypes of black-grass (Alopecurus myosuroidesHuds.) to the herbicide, chlorotoluron, was characterized inglasshouse and laboratory studies. ED₅₀values, defined as theamount (kg active ingredient ha^-1) of chlorotoluron requiredto reduce fresh mass by 50% under standard conditions, weredetermined for a resistant biotype (39.3 kg a.i. ha^-1) collectedfrom Peldon, Essex, UK and a susceptible biotype (0.93 kg a.i.ha^-1) obtained commercially, giving a resistance factor of 42.The resistance factor was calculated as the ratio of ED₅₀valuesand describes the increase in amount of herbicide needed toreduce fresh mass by 50% in the resistant, compared to the susceptible,biotype. Resistance was further characterized by measurementsof whole plant growth and photosynthesis. Relative growth rate,number of tillers, leaf area and mean fresh mass were the samein untreated plants of both biotypes, and rates of photosynthesisat both high and low photon flux were similar, with no differencein apparent quantum yield. Photosynthesis by whole plants wasstudied over a 24 h period following chlorotoluron treatment.Resistant plants showed no reduction in photosynthesis overthis period, whereas photosynthesis by susceptible plants ceased10 h after treatment and did not recover. Alopecurus myosuroides ; black-grass; herbicide resistance; chlorotoluron     

Mechanism of isoproturon resistance in <Emphasis Type="Italic">Phalaris minor:</Emphasis> in silico design,synthesis and testing of some novel herbicides for regaining sensitivity

Isoproturon, 3-p-cumenyl-1 dimethylurea was the only herbicide controlling Phalaris minor, a major weed growing in wheat fields till the early 1980s. Since it has acquired resistance against isoproturon, like other substituted urea herbicides, where the identified target site for isoproturon is in the photosynthetic apparatus at D1 protein of Photosystem-II (PS-II). Nucleotide sequence of susceptible and resistant psbA gene of P. minor has been reported to have four point mutations. During the present work D1 protein of both susceptible and resistant biotypes of P Minor has been modeled. Transmembrane segments of amino acids were predicted by comparing with the nearest homolog of bacterial D1 protein. Volume and area of active site of both susceptible and resistant biotypes has been simulated. Isoproturon was docked at the active site of both, susceptible and resistant D1 proteins. Modeling and simulation of resistance D1 protein indicates that the resistance is due to alteration in secondary structure near the binding site, resulting in loss in cavity area, volume and change in binding position, loss of hydrogen bonds, hydrophobic interaction and complete loss of hydrophobic sites. To regain sensitivity in resistant biotype new derivatives of isoproturon molecules have been proposed, synthesized and tested. Among the 17 derivatives we found that the N-methyl triazole substituted isoproturon is a potential substitute for isoproturon.     

Sensitivity of Eleusine indica Callus to Trifluralin and Amiprophosmethyl in Correlation with the Binding of These Compounds to Tubulin

The sensitivity of calluses derived from susceptible and resistant goosegrass (Eleusine indica (L.) Gaertn.) biotypes to dinitroaniline herbicides, which disrupt interphase and mitotic-spindle microtubules, was evaluated. A callus culture derived from the resistant biotype retained resistance to both trifluralin (dinitroaniline herbicide) and amiprophosmethyl (phosphorothioamidate herbicide). The site for the interaction between -tubulin subunit and dinitroaniline or phosphorothioamidate herbicides was identified by computer simulation. A correlation was found between the level of callus sensitivity to herbicide tested and the pattern of herbicide interaction with -tubulin.     

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.     

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.     

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.     

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.     

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.     

Mode of action studies on nitrodiphenyl ether herbicides: I. Use of barley mutants to probe the role of photosynthetic electron transport

5-[2-Chloro-4-(trifluoromethyl)phenoxy]-2-nitroacetophenone oxime-o-(acetic acid, methyl ester) (DPEI), is a potent nitrodiphenyl ether herbicide which causes rapid leaf wilting, membrane lipid peroxidation, and chlorophyll destruction in a process which is both light- and O₂-dependent. These effects resemble those of other nitrodiphenyl ether herbicides. Unlike paraquat, the herbicidal effects of DPEI are only slightly reduced by pretreatment with the photosynthetic electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea. DPEI is a weak inhibitor of photosynthetic electron transport (I₅₀ 15 micromolar for water to paraquat) in vitro, with at least one site of action at the cytochrome b₆f complex. Ultrastructural studies and measurements of ethane formation resulting from lipid peroxidation indicate that mutants of barley lacking photosystem I (PSI) (viridis-zb⁶³) or photosystem II (viridis-zd⁶⁹) are resistant to paraquat but susceptible to DPEI. The results indicate that electron transfer through both photosystems is not essential for the toxic effects of nitrodiphenyl ether herbicides. Furthermore, the results show that neither cyclic electron transport around PSI, nor the diversion of electrons from PSI to O₂ when NADPH consumption is blocked are essential for the phytotoxicity of nitrodiphenyl ether herbicides.