Glyphosate tolerance by Clitoria ternatea and Neonotonia wightii plants involves differential absorption and translocation of the herbicide

Glyphosate tolerance by Clitoria ternatea, Neonotonia wightii and Amaranthus hybridus was studied in whole plants from Mexico. Experiments in a controlled growth chamber showed both legumes to be highly tolerant of glyphosate, with and ED₅₀ values of 600.18 g ae ha^?C1 for C. ternatea and 362.94 g ae ha^?C1 for N. wightii. On the other hand, A. hybridus was highly susceptible to the herbicide (ED₅₀?=?42.22 g ae ha^?C1). Shikimate accumulation peaked 96 h after treatment in the tolerant plants and the susceptible weed under 500 g ae ha^?C1 glyphosate. The shikimic acid content of whole leaves was 4.0 and 5.0 times higher in the susceptible weed than in N. wightii and C. ternatea, respectively. ¹⁴C-glyphosate absorption and translocation tests showed A. hybridus to absorb 30% more herbicide than the legumes 24 h after glyphosate foliar application. ¹⁴C-glyphosate translocation as measured by quantified autoradiography revealed increased translocation of the herbicide to untreated leaves and roots in A. hybridus relative to the two legumes. The cuticular surface of A. hybridus exhibited very low wax coverage relative to the epicuticular surface of N. wightii and, especially, C. ternatea. No significant degradation of glyphosate to aminomethylphosphonic acid and glyoxylate metabolites was detected among the tolerant leguminous plants or the susceptible weed population. These results indicate that the high glyphosate tolerance of Clitoria ternatea and Neonotonia wightii is mainly a result of poor penetration and translocation of the herbicide to apical growing points in their plants.     

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.     

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.     

Effects of glyphosate on the movement of assimilates of two Lolium perenne L. populations with differential herbicide sensitivity

Glyphosate applications trigger the depletion of aromatic amino acid pools and the decrease of photosynthesis that results in changes in carbon metabolism. The aim of this work was to determine the effect of glyphosate on the export of ¹⁴C from ¹⁴C-glucose to the main sinks, by comparing a glyphosate-resistant Lolium perenne population with a susceptible one. Untreated plants of the two populations grown in hydroponics were labeled with ¹⁴C-glucose applied at the youngest expanded leaf at the tillering stage. Similar ¹⁴C-glucose absorption and ¹⁴C distribution patterns were recorded in both populations. In another experiment, half of the plants of each population were treated with glyphosate, whereas the other half was sprayed with water (controls). Glucose absorption did not vary under glyphosate treatment, regardless of the sensitivity of each population to the herbicide. However, the translocation of ¹⁴C and its distribution patterns were significantly affected by glyphosate within 1 day in the susceptible population. The treated susceptible plants showed 57% higher ¹⁴C retention at the labeled area than their controls. The lower ¹⁴C movement significantly affected the unexpanded leaves and the apical meristem on the labeled tiller. Moreover, the ¹⁴C released from roots was significantly decreased by glyphosate only in the susceptible plants. Glyphosate did not influence leaf absorption, translocation, or release of ¹⁴C-labeled glucose plus radiolabeled metabolites in the resistant population.     

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.     

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.     

Distinct non-target site mechanisms endow resistance to glyphosate,ACCase and ALS-inhibiting herbicides in multiple herbicide-resistant <Emphasis Type="Italic">Lolium rigidum</Emphasis>

This study investigates mechanisms of multiple resistance to glyphosate, acetyl-coenzyme A carboxylase (ACCase) and acetolactate synthase (ALS)-inhibiting herbicides in two Lolium rigidum populations from Australia. When treated with glyphosate, susceptible (S) plants accumulated 4- to 6-fold more shikimic acid than resistant (R) plants. The resistant plants did not have the known glyphosate resistance endowing mutation of 5-enolpyruvylshikimate-3 phosphate synthase (EPSPS) at Pro-106, nor was there over-expression of EPSPS in either of the R populations. However, [¹⁴C]-glyphosate translocation experiments showed that the R plants in both populations have altered glyphosate translocation patterns compared to the S plants. The R plants showed much less glyphosate translocation to untreated young leaves, but more to the treated leaf tip, than did the S plants. Sequencing of the carboxyl transferase domain of the plastidic ACCase gene revealed no resistance endowing amino acid substitutions in the two R populations, and the ALS in vitro inhibition assay demonstrated herbicide-sensitive ALS in the ALS R population (WALR70). By using the cytochrome P450 inhibitor malathion and amitrole with ALS and ACCase herbicides, respectively, we showed that malathion reverses chlorsulfuron resistance and amitrole reverses diclofop resistance in the R population examined. Therefore, we conclude that multiple glyphosate, ACCase and ALS herbicide resistance in the two R populations is due to the presence of distinct non-target site based resistance mechanisms for each herbicide. Glyphosate resistance is due to reduced rates of glyphosate translocation, and resistance to ACCase and ALS herbicides is likely due to enhanced herbicide metabolism involving different cytochrome P450 enzymes.     

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.     

Genetic mapping of non-target-site resistance to a sulfonylurea herbicide (Envoke®) in Upland cotton (Gossypium hirsutum L.)

Acetolactate synthase (ALS) is responsible for a rate-limiting step in the synthesis of essential branched-chain amino acids. Resistance to ALS-inhibiting herbicides, such as trifloxysulfuron sodium (Envoke^®), can be due to mutations in the target gene itself. Alternatively, plants may exhibit herbicide tolerance through reduced uptake and translocation or increased metabolism of the herbicide. The diverse family of cytochrome P450 proteins has been suggested to be a source of novel herbicide metabolism in both weed and crop plants. In this study we generated a mapping population between resistant and susceptible cotton (Gossypium hirsutum L.) cultivars. We found that both cultivars possess identical and sensitive ALS sequences; however, the segregation of resistance in the F2 progeny was consistent with a single dominant gene. Here we report the closely linked genetic markers and approximate physical location on chromosome 20 of the source of Envoke herbicide susceptibility in the cotton cultivar Paymaster HS26. There are no P450 proteins in the corresponding region of the G. raimondii Ulbr. genome, suggesting that an uncharacterized molecular mechanism is responsible for Envoke herbicide tolerance in G. hirsutum. Identification of this genetic mechanism will provide new opportunities for exploiting sulfonylurea herbicides for management of both weeds and crop plants.     

Translocation and Complex Formation of Picloram and 2,4-D in Rape and Sunflower

Uptake, translocation and complex formation of ¹⁴C-labelled 4-amino-3,5,6-trichloropicolinic acid (picloram) and 2,4-dichlorophenoxyacetic acid (2,4-D) in seedlings of rape (Brassica napus L. cv. Nilla) and sunflower (Helianthus annuus L. var. uniflorus) were studied. Sunflower is susceptible both to 2,4-D and picloram, while rape is susceptible to 2,4-D but more tolerant to picloram. The uptake of the herbicides through the leaves was almost complete in both species. Translocation of 2,4-D into the roots took place more readily than that of picloram. In sunflower about 50 per cent of the applied 2,4-D was extruded through the roots into the nutrient solution after 9 days. In the picloram-treated sunflower most of the activity was found in the aerial parts, while in picloram-treated rape most of the activity still occurred in the treated leaf after 9 days. No activity at all was found in the roots or in the nutrient solution of the picloram-treated rape seedlings. While the major part of 2,4-D always was found in the state of free herbicide, a large fraction of picloram was rapidly bound into water-soluble complexes. This binding was especially pronounced in rape. Separation by paper chromatography showed that different radioactive compounds were formed. Most of these could be hydrolyzed, thereby releasing free herbicide. The results support the hypotheses that complex formation could counteract herbicide translocation and toxicity of auxin herbicides.     

Absorption,translocation, and metabolism of flurtamone in sicklepod (Cassia obtusifolia) and peanut (Arachis hypogea)

The basis of peanut tolerance to the bleaching herbicide flurtamone was examined. The absorption, translocation, and metabolism of ¹⁴C-flurtamone were examined in peanut at 6, 24, and 48 h after root application. Differences in ¹⁴C-flurtamone uptake over time were not detected. Approximately 40% of the absorbed ¹⁴C-flurtamone was in the leaves at 6 h after treatment; 60% was metabolized to polar products 41% of absorbed ¹⁴C in 6 h; 40% of this moved from roots to shoots; and 60% of this did not co-chromatograph with the parent; 9.8% of applied ¹⁴C-flurtamone was altered in leaf tissue. The levels of metabolized flurtamone increased with time after treatment (75% and 83% of applied ¹⁴C-flurtamone metabolized at 24 and 48 h, respectively). Parent ¹⁴C-flurtamone was detectable with R_t of 7 min and unknown metabolites with an R_t of 3.3, 4.4, and 5.6 min, respectively, was detected in leaf tissue at 6, 24, and 48 h after treatment.     

Uptake and Translocation of Benthiocarb Herbicide by Plants

The uptake and translocation of ¹⁴C-benthiocarb labelled at benzyl methylene by rice plant, bamyardgrass, wild amaranth, smart weed and lambsquarters were investigated, ¹⁴C-Benthiocarb was absorbed through the roots and the radioactivity was translocated into whole plants. The rate of absorption and translocation varied by the kind of plants. The translocation was occurred not only from roots into leaves, but from a leaf into other leaves, and even into roots of some kinds of plant. The absorption and translocation was more easy in barnyard-grass than in rice plant. Benthiocarb was rapidly absorbed by seeds and accumulated mostly in the embryo. The uptake of benthiocarb by seedlings decreased with the order of mesocotyl (bamyardgrass only), coleoptyl, root and leaf. Benthiocarb was degraded rapidly in plants.     

Ameliorative effects of squash (<Emphasis Type="Italic">Cucurbita moschata</Emphasis> Duchesne ex Poiret) leaf extracts on oxidative stress

This study screened paraquat-tolerant plants among 10 plant species, including monocots and dicots angiosperms. Squash (Cucurbita moschata Duchesne ex Poiret) and kidney bean (Phaseolus vulgaris L.) plants exhibited the highest photooxidation-tolerant phenotypes upon a foliar treatment with paraquat. A foliar treatment with paraquat pre-mixed with leaf water extracts from the squash plant significantly alleviated paraquat-induced oxidative damage in maize, but this was not the case after a treatment with the hydrophobic phase of the leaf extracts. In particular, the water extract from young leaves (4th true leaf) of squash plants conferred tenfold higher tolerance to oxidative damage in paraquat-treated leave tissues compared to paraquat-only treatment. This tolerance was tightly linked not only to the increased amounts of ascorbic acid and dehydroascorbate antioxidants in the damaged leaves, but also to the reduced chlorophyll loss, lipid peroxidation, and cellular electrolyte leakage. Moreover, the protective effects of the water extract were apparent when using another bipyridyl herbicide, diquat, but not with a diphenyl-ether herbicide, oxyfluorfen. On the other hand, pre-treatment with the extract prior to the onset of drought or cold stress had no significant antioxidative effect on the treated tissues.     

Oxidative stress tolerance and longevity in Arabidopsis: the late‐flowering mutant gigantea is tolerant to paraquat

Recent genetic analyses of longevity in animals have revealed that long-lived strains are more tolerant to environmental stresses. To investigate whether extended longevity in Arabidopsis also correlates with an increase in stress tolerance, the response was tested of 11 late-flowering mutants to the superoxide radical-generating herbicide paraquat. A tight correlation between flowering time and paraquat tolerance was found when plants were exposed to low doses of herbicide. Furthermore, the mutant gigantea (gi-3) with the longest delay in flowering time had a high tolerance level to paraquat-induced oxidative stress. All the tested gi alleles had an increased tolerance to paraquat toxicity compared to wild-type, although the actual levels of tolerance differed. In addition, the gi-3 mutant was more tolerant to hydrogen peroxide. These results suggest that the link between longevity and oxidative stress resistance in plants is similar to that found in animals, implying that this phenomenon may be general for all aerobic organisms.     

Limited uptake, translocation and enhanced metabolic degradation contribute to glyphosate tolerance in Mucuna pruriens var. utilis plants

Velvet bean (Mucuna pruriens, Fabaceae) plants exhibits an innate, very high resistance (i.e., tolerance) to glyphosate similar to that of plants which have acquired resistance to this herbicide as a trait. We analyzed the uptake of [(14)C]-glyphosate by leaves and its translocation to meristematic tissues, and used scanning electron micrographs to further analyze the cuticle and 3D capillary electrophoresis to investigate a putative metabolism capable of degrading the herbicide. Velvet bean exhibited limited uptake of glyphosate and impaired translocation of the compound to meristematic tissues. Also, for the first time in a higher plant, two concurrent pathways capable of degrading glyphosate to AMPA, Pi, glyoxylate, sarcosine and formaldehyde as end products were identified. Based on the results, the innate tolerance of velvet bean to glyphosate is possibly a result of the combined action of the previous three traits, namely: limited uptake, impaired translocation and enhanced degradation.     

Selection of Glyphosate-Tolerant Tobacco Calli and the Expression of this Tolerance in Regenerated Plants

From nonmutagenized haploid suspensions of Nicotiana tabacum L. cv Wisconsin 38 cells, 51 cell lines capable of growth in the presence of 1 millimolar glyphosate (N-phosphonomethyl glycine) were initially isolated at a frequency of 2.3 × 10⁻⁸. Eighteen cell lines retained tolerance when grown on selective medium for 3 years. Tolerance persisted for at least 14 months in six cell lines cultured in the absence of glyphosate. Some plants regenerated from four glyphosate-tolerant cell lines were tolerant. Glyphosate-tolerant tissue was isolated from some sensitive as well as some tolerant regenerated plants. Six of the tolerant cell lines were also tolerant to the herbicide amitrole (3-amino-1,2,4-triazole). Five cell lines selected for amitrole tolerance were glyphosate tolerant. Some plants regenerated from three of these five cell lines were glyphosate tolerant and glyphosate-tolerant tissue was obtained from several of these regenerated plants. Amitrole uptake in suspension cultures of several variants was assessed in terms of influx rate constants. This parameter was not sufficiently different indicating that altered membrane properties could not account for the herbicide tolerance.     

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.     

The relationship between the rate of entry of ioxynil and effects on photosynthesis in normal and drought-stressed Stellaria media

The entry of ¹⁴C-labelled ioxynil octanoate into leaves of Stellaria media has been measured for plants grown in dry or moist soil. Of the total herbicide applied, 1–3% entered the leaf by 24 h and 2–5% by 72 h after treatment. Entry into moist-grown plants proceeded at about twice the rate of that into drought-stressed plants. Despite the limited rate of entry, the inhibitory action of ioxynil octanoate on photosynthetic carbon dioxide exchange was rapid, inhibition within 24 h reaching 70–90% in moist-grown plants, and 30–70% in dry-grown plants. Plants grown under moisture stress contained greater concentrations of the pigments chlorophyll a, carotene and lutein (a xanthophyll) than did moist-grown plants, and ioxynil-induced breakdown of these pigments was more rapid in moist-grown plants. It is suggested that these factors contribute to the greater tolerance of drought-stressed S. media to applications of ioxynil octanoate. The importance of continuous measurements of herbicide action is discussed in relation to the value and interpretation of ¹⁴C uptake data.